This work was supported by the Austrian Science Fund (FWF grants P23797, P27275 and P30596) and the Swiss National Science Foundation (Grant #PBBEP2_146174).

<ol>
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The authors certify they have no affiliation with any for-profit organization.

Solid-phase DNA and RNA synthesis is the bread and butter of every nucleic acid chemistry lab, and although the addition of the photolithography component is admittedly a complex operation, microarray fabrication mediated by UV light is also a very reliable process. It is, in addition, the only available method for in situ RNA synthesis on microarrays. Still, as in any multi-stage experimental procedure, there is ample room for human error.
Perhaps the most critical step is the coupling of a phosphoramidite, as it needs to be a constantly high-yielding chemical reaction in order to afford oligonucleotides with few synthetic errors. In our microarray synthesis protocol, phosphoramidite coupling is even more pivotal to overall synthesis quality since the fabrication process bypasses capping and prevents oligonucleotide purification. Stepwise coupling efficiencies above 99% have been calculated for all photosensitive DNA and RNA phosphoramidites, even for very short coupling times (15 s)24 but lower coupling yields can occasionally occur, particularly in the case of dG amidites. The stability of the solubilized phosphoramidites at room temperature has been investigated before and was shown to depend on the nature of the nucleobase, with guanosine phosphoramidites prone to extensive degradation in only a matter of days29,30. But when stored at -25 &#176;C, dG phosphoramidites dissolved in ACN as 30 mM solution were found to be stable for several weeks. The relative instability of dG phosphoramidite solutions at room temperature does however mean that they should not be kept attached to the DNA synthesizer for several days.
For RNA phosphoramidites, the coupling yield is very dependent on phosphoramidite quality (which can be assessed by 31P NMR spectroscopy) and coupling time. Coupling times of 5 minutes for rA, rG, rC and 2 min for rU appear necessary. Indeed, we found that shortening the condensation time to 2 min for all RNA phosphoramidites led to significantly lower hybridization signals.
The DNA synthesizer itself, as well as the reagents and solvents, certainly needs to be as clean as possible in order to achieve the highest yield of oligonucleotide synthesis. However, insoluble material, salts or particles, can accumulate over time in the lines and tubing of the delivery system, leading to a gradual decrease in consumption of reagents and reactants. Where a general cleaning of the synthesizer does not resolve a low output volume, an increase in the number of pulses can be an alternative solution. Particularly useful in the case of low phosphoramidite consumption, the line in the coupling protocol corresponding to the pumping of a mix of phosphoramidite and activator (third line of the coupling subsection in Table 1 and Table 2) can be modified, from 6 to 9 pulses without any appreciable negative effect on synthesis quality. Furthermore, the number of pulses of activator needed to bring the amidite/activator mix to the synthesis substrate (currently 6, fourth line in the coupling subsection, see Table 1 and Table 2) depends on the DNA synthesizer itself as well as on tubing length in the synthesis cell. This number can be adjusted after replacing the phosphoramidite with a colored solution and counting the number of pulses needed to push the colored mix to the glass substrate for coupling.
The method described herein allows for DNA and RNA synthesis to proceed simultaneously, on the same microarray. Hybrids of DNA and RNA may also be prepared without any change to the array fabrication protocols, and as long as the three-step deprotection protocol is followed. However, it should be noted that RNA-only microarrays only require a two-step deprotection: a decyanoethylation first with Et3N followed by hydroxyl and base deprotection with hydrazine. DNA nucleobases were found to be incompletely deprotected under those conditions, and need the additional step in EDA in order to effect the complete removal of phenoxyacetyl (Pac) groups. This extra treatment with EDA is shorter (5 min) than for the standard deprotection of DNA microarrays31, but it is sufficient to drive it to completion after the triethylamine and hydrazine treatments. In addition, a short reaction time with EDA limits the exposure of a fully-deprotected RNA oligonucleotide to basic conditions.&#160;&#160;
An advantage of in situ RNA array synthesis over alternative methods like spotting or DNA transcription32,33,34 is the ability to store the synthesized RNA microchip in protected form until use, thus avoiding the risk of potential RNA degradation. Post-synthetic procedures for RNA does, on the other hand, imply that consumables and reagents are kept sterile and that handling is performed under RNase-free conditions. Of note, we found that the addition of RNase inhibitor to the hybridization mix did not yield stronger hybridization signals for the RNA features.
The synthesis of DNA libraries on a base-sensitive monomer is more complex than the synthesis of a few control sequences on a surface, and as such is certainly more prone to design errors. Yet, assuming that the sequence design (i.e., the nature and the number of sequences) is correct, transforming this list into a collection of exposure masks and an ordered series of coupling cycles remains a straightforward process. However, important variations from standard microarray synthesis exist and are critical to a successful fabrication of a high-density library array.
First, a base-sensitive dT monomer is coupled as the first phosphoramidite after synthesis of the linker. The coupling yield of this monomer (Figure 1) was found to be relatively low, around 85%28, which is why efforts are made to improve its incorporation rate, either by increasing its concentration in ACN from 30 mM to 50 mM, or by repeating the coupling step: two consecutive coupling reactions using fresh monomers, or two separate but consecutive coupling cycles.
The second change is the addition of a &#946;-carotene solution in the back chamber of the synthesis cell, which conveniently absorbs 365 nm light. This is an important modification of the photolithography setup as it prevents UV light from reflecting back onto the array substrate. Indeed, after traversing the interstitial medium between the substrates, incoming UV light exits through the drilled slide and reaches the quartz block of the cell. The Fresnel equations predict that ~4% of perpendicularly incident UV light will reflect from each of the three downstream air-glass interfaces (exit side of the 2nd substrate and both sides of the quartz block) and back onto the synthesis substrate, leading to unintended exposure of photoprotected oligonucleotides. Diffraction and scattering also contribute to &#34;off-target&#34;&#160;photodeprotection and, therefore, to nucleotide insertion, which directly affects the error rate of synthesis, but these contributions are much smaller than reflections and can mainly be addressed by reducing synthesis density (leaving gaps between features). We have found that the level of &#946;-carotene solution in the lower chamber of the cell tends to slightly decrease only during the first minutes of array synthesis, and therefore needs to be monitored and readjusted.
Finally, the third change is the deprotection solution, replacing EtOH for toluene, which keeps the cleaved DNA library bound to the surface, presumably through electrostatic interactions. Applying a small amount of water to the synthesis area after ACN washing allows for the library to be conveniently collected. The process is however only successful if the water content in EDA and toluene is minimal, rendering the nucleic acid entirely insoluble in the deprotection cocktail. Alternatively, DNA libraries may be cleaved off the chip using ammonia9,10,14,35, then further deprotected by heating the DNA-containing aqueous ammonia solution to 55 &#176;C overnight. The recovery of DNA libraries using ammonia is however not compatible with RNA. RNA oligonucleotides on a base-cleavable&#160;substrate can be eluted from the surface using the same EDA/toluene procedure described above, but only at the penultimate stage after the Et3N and hydrazine two-step deprotection strategy28.
Alternative strategies to recover oligonucleotide pools from microarrays without the need for a specific basic treatment exist, are in principle compatible with photolithography and rely on the use of enzymes. For instance, a single deoxyuracil nucleotide can be the target of the uracil-DNA glycosylase (UDG) and excised from the rest of the DNA sequence, or a single RNA unit can be recognized by RNase H type 2 enzymes and the phosphodiester bond 5&#39;&#160;to the RNA cleaved, releasing the 5&#39;&#160;DNA part23.
We now have a powerful, reliable and high-density method for the synthesis of DNA, RNA, and hybrid DNA/RNA microarrays. These can not only serve as platforms for hybridization or binding assays36, but they also represent a fast and inexpensive way to produce complex nucleic acid libraries. For DNA-based digital data storage, microarray photolithography may become a potential solution to the &#34;writing&#34;&#160;bottleneck (i.e., to the encoding of information by synthesis). The success in digital encoding on DNA and in de novo gene assembly depends on sequence fidelity which, at the synthesis level, translates into the error rate. Synthetic and optical errors in our current array fabrication protocols will be discussed and reported on elsewhere. In parallel, efforts are now underway to further increase fabrication scale and throughput.

The practical use of DNA microarrays have traditionally been in the study of the variations in the gene expression levels between two cell populations, using complementary strands and fluorescence as a detection method1. Occasionally, DNA microarrays venture into binding events with non-nucleic acid ligands, such as proteins, with a strategy of systematic sequence permutation that offers a comprehensive overview of the binding landscape2,3,4,5. This approach effectively transforms microarrays from mere hybridization surfaces into platforms with broad sequence coverage, which would be an asset for the study of the richer and more complex world of RNA structure and function. Supported by the extremely efficient phosphoramidite coupling reaction6, in situ synthesized DNA arrays can now also be regarded as a cheap source of DNA7, which is becoming particularly relevant considering the ever-increasing demand for nucleic acid material for gene assembly8,9, DNA-based nanostructures10, information storage or sequencing11,12. Likewise, sequencing technologies are likely to benefit from the development of methods that yield very complex mixtures of RNA oligonucleotides13. In this context, array fabrication protocols that allow for oligonucleotides to be synthesized in situ and at high density are ideally placed to meet the needs of the rapidly expanding field of nucleic acid biotechnology. However, with a field as diverse as biotechnology, the purpose of each application may require that DNA on microarray be produced either at high-throughput or with a very low amount of synthetic errors14,15, or both, requiring a closer look at the synthesis protocols of DNA microarrays which, historically, have been primarily optimized for hybridization assays. Meanwhile, in situ synthesis of RNA microarrays has been found to be a challenging endeavor, with most of the difficulty associated with the protecting group for the 2&#39;-OH function, usually a silyl moiety in standard solid-phase synthesis that is removed with fluorine-based reagents, chemicals that are incompatible with glass or silicon surfaces. Those issues and challenges in DNA and RNA microarray synthesis have lately been the subject of a large body of work, in particular with the photolithography approach16.
Photolithography uses UV light to unblock oligonucleotides before coupling and requires masks to construct a pattern of UV exposure, thereby spatially organizing and controlling the growth of oligonucleotides. Physical masks have been replaced by computer-controlled micromirrors&#160; whose tilting selectively reflects UV light onto the microarray substrate17,18,19. As a UV source, we use 365 nm light from a high power LED source20. Current photolithographic setups are equipped with micromirror arrays containing 1024 &#215; 768 mirrors, corresponding to more than 780,000 individually addressable spots (&#34;features&#34;) on a small area of just 1.4 cm2, or 1080p with arrays of 1920 &#215; 1080, or &#62;2 million mirrors. Each of the mirrors in the device therefore has direct control over the sequence grown on the corresponding feature. With the exception of UV light, photolithography functions like a solid-phase synthesis technique and adopts the cycle-based phosphoramidite chemistry. Only it requires an entirely different protection strategy for RNA synthesis to succeed. We developed a new series of light-sensitive RNA phosphoramidites bearing hydrazine-labile protecting groups21. These monomers allow for the RNA to be deprotected under mild conditions that do not affect the integrity of the surface. A first deprotection step uses triethylamine to remove the cyanoethyl phosphodiester protecting groups, while hydrazine is used in a second, separate step to remove those at the 2&#39;-OH and exocyclic amine functions. In so doing, RNA oligonucleotides ~30-nt in length and of any sequence can now be synthesized in situ on microarrays22,23. In parallel, we also recently started to address the questions of throughput, quality and speed in DNA and RNA photolithography. We measured coupling efficiencies &#62;99% for all DNA and RNA amidites (Figure 1) and investigated each individual step in the oligonucleotide elongation cycle, from oxidation time, to choice of activator and to optimal UV exposure24,25. We have brought in new light-sensitive 5&#39; protecting groups that can be removed in seconds only, transforming the synthesis of hundreds of thousands of 100mers into a few hours-long process26. We have also doubled the throughput of array fabrication by exposing two substrates simultaneously27. Finally, we have introduced a dT phosphoramidite containing a base-sensitive succinyl group as a convenient way to cleave, collect and analyze DNA and RNA oligonucleotides, which is central to library preparation28.
In spite of the relatively mundane aspect of DNA and RNA solid-phase synthesis, especially for nucleic acid chemists, microarray photolithography remains a non-trivial upgrade requiring a complex setup, careful control and supervision of the process, and separate instructions for post-synthetic handling depending on the nature of the oligonucleotide and the type of application. In this article, we wish to provide a detailed presentation of the entire step-by-step procedure of in situ synthesis of DNA and RNA microarrays by photolithography, from experimental design to data analysis, with an emphasis on the preparation of instruments and consumables. We then describe the post-synthetic deprotection methods that correspond to the intended purpose of microarray fabrication (i.e., either hybridization or the recovery of nucleic acid libraries).&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:792px;" width="792">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Slide functionalization</td>
			<td style="width:175px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Acetic acid &#62;99.8%</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">33209</td>
			<td>For RNA deprotection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">CNC router</td>
			<td style="width:175px;">Stepcraft</td>
			<td style="width:119px;">300 CK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Ethanol absolute</td>
			<td style="width:175px;">VWR</td>
			<td style="width:119px;">1.07017.2511</td>
			<td>For deprotection and functionalization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">N-(3-triethoxysilylpropyl)-4-hydroxybutyramide</td>
			<td style="width:175px;">Gelest</td>
			<td style="width:119px;">SIT8189.5</td>
			<td>Silanizing reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Nexterion Glass D microscope slides</td>
			<td style="width:175px;">Schott</td>
			<td style="width:119px;">1095568</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Polymax 1040</td>
			<td style="width:175px;">Heidolph</td>
			<td style="width:119px;"></td>
			<td>Orbital shaker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Proclean 507 Ultrasonic water bath</td>
			<td style="width:175px;">Ulsonix</td>
			<td style="width:119px;"></td>
			<td>To clean slides after drilling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Tickopur RW 77 Special Purpose Cleaner</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">Z860086</td>
			<td>To clean slides after drilling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Microarray synthesis</td>
			<td style="width:175px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">0.25 M dicyanoimidazole in ACN</td>
			<td style="width:175px;">Biosolve</td>
			<td style="width:119px;">0004712402BS</td>
			<td>Activator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">0.7 XGA DMD</td>
			<td style="width:175px;">Texas Instruments</td>
			<td style="width:119px;"></td>
			<td>Digital Micromirror Device</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:332px;">20 mM I2 in pyridine/H2O/THF</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">L860020-06</td>
			<td>Oxidizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">250 &#956;m thick Chemraz 584 perfluoroelastomer</td>
			<td style="width:175px;">FFKM</td>
			<td style="width:119px;"></td>
			<td>Lower teflon gasket</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">2&#39;-O-ALE RNA phosphoramidites</td>
			<td style="width:175px;">ChemGenes</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">365 nm high-power UV-LED</td>
			<td style="width:175px;">Nichia</td>
			<td style="width:119px;">NVSU333A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">5&#39;BzNPPOC DNA phosphoramidites</td>
			<td style="width:175px;">Orgentis</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">5&#39;NPPOC DNA phosphoramidites</td>
			<td style="width:175px;">FlexGen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Acetonitrile</td>
			<td style="width:175px;">Biosolve</td>
			<td style="width:119px;">0001205402BS</td>
			<td>For DNA synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Amidite Diluent for DNA synthesis</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">L010010</td>
			<td>For dissolving phosphoramidites</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Cleavable dT</td>
			<td style="width:175px;">ChemGenes</td>
			<td style="width:119px;"></td>
			<td>Base-sensitive monomer for library preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">DMSO</td>
			<td style="width:175px;">Biosolve</td>
			<td style="width:119px;">0004474701BS</td>
			<td>As exposure solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">DNA and RNA microarray deprotection</td>
			<td style="width:175px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Ethylenediamine &#62;99.5%</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">3550</td>
			<td>For deprotection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Expedite 8909</td>
			<td style="width:175px;">PerSeptive Biosystems</td>
			<td style="width:119px;"></td>
			<td>DNA synthesizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Hydrazine hydrate 50-60% hydrazine</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">225819</td>
			<td>For RNA deprotection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Imidazole</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">56750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Industrial Strength lower-density PTFE tape</td>
			<td style="width:175px;">Gasoila</td>
			<td style="width:119px;"></td>
			<td>Thin, upper teflon gasket</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Pyridine &#62;99%</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">P57506</td>
			<td>For RNA deprotection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Triethylamine &#62;99%</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">T0886</td>
			<td>For RNA deprotection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">&#946;-carotene</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">C9750</td>
			<td>For library preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Hybridization and scanning</td>
			<td style="width:175px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">20x Sodium Saline Citrate</td>
			<td style="width:175px;">Roth</td>
			<td style="width:119px;">1054.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">5&#39;Cy3-labelled complementary strand</td>
			<td style="width:175px;">Eurogentec</td>
			<td style="width:119px;"></td>
			<td>For duplex hybridization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Biopur Safe-Lock microcentrifuge tube</td>
			<td style="width:175px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">BSA (10 mg/mL)</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">R3961</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">EDTA molecular biology grade</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">H5031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">GenePix 4100A</td>
			<td style="width:175px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td>Microarray scanner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Hybridization oven</td>
			<td style="width:175px;">Boekel Scientific</td>
			<td style="width:119px;">230500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">MES monohydrate</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">69889</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">MES sodium</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">M3058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">NaCl &#62;99.5%</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">71376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">SecureSeal SA200 hybridization chamber</td>
			<td style="width:175px;">Grace BioLabs</td>
			<td style="width:119px;">623503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Spectrafuge mini</td>
			<td style="width:175px;">Labnet</td>
			<td style="width:119px;">C1301</td>
			<td>Microarray centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Tween-20 molecular biology grade</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">P9416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Data extraction</td>
			<td style="width:175px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Excel</td>
			<td style="width:175px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td>For data extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">MatLab</td>
			<td style="width:175px;">MathWorks</td>
			<td style="width:119px;"></td>
			<td>Microarray design</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">NimbleScan 2.1</td>
			<td style="width:175px;">Roche NimbleGen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Desalting and quantification</td>
			<td style="width:175px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">NanoDrop One Spectrophotometer</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Toluene</td>
			<td style="width:175px;">Merck</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">ZipTip C18</td>
			<td style="width:175px;">Millipore</td>
			<td style="width:119px;">ZTC18s008</td>
			<td>Desalting pipet tips</td>
		</tr>
	</tbody>
</table>

high-density dna and rna microarrays - photolithographic synthesis, hybridization and preparation of large nucleic acid libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

DNA and RNA microarray hybridization 
Figure 5 shows the results of a hybridization assay performed on a microarray containing the DNA and RNA versions of a 25mer sequence (5&#39;-GTCATCATCATGAACCACCCTGGTC-3&#39;&#160;in DNA form). The scan in Figure 5A appears in a greenscale format corresponding to the excitation/emission spectrum of Cy3 fluorescence, with fluorescence intensity recorded in arbitrary units between 0 and 65536. The array design followed the 25:36 feature layout described in the protocol section. The scan is shown after proper orientation of the array, with the top left corner populated with the longest chain of fiducial features. Here, fiducial features contain the DNA version of the 25mer and should, in principle, always give a positive fluorescence signal in order to perform scan alignment and data extraction. The hybridized microarray should appear uniformly bright, with the edges of the synthesis area being however usually brighter than the center (up to 50% brighter). The large amount of sequence replicates, randomly distributed throughout the area, reduces the impact of spatial artefacts. Here, each sequence (DNA and RNA) was synthesized at 2,000 random locations. There is typically low fluorescence noise (background), &#60;50 a.u., which leads to a signal/noise ratio in the order of 200:1 to 800:1 in hybridization assays. After data extraction, fluorescence intensities are averaged out and plotted &#177;SD.
There is significant variability in absolute fluorescence values between experiments. Here, we show the results for three independent syntheses using the same fabrication parameters and the same post-synthetic handling. The 25mer DNA, when hybridized to its complementary Cy3-labelled DNA strand, will yield fluorescence signals ranging anywhere from 20,000 to 30,000, very rarely above or below. The 25mer RNA, when hybridized to the same Cy3-labelled DNA complement, will give fluorescence intensities on the corresponding features ranging from 15,000 to 20,000. However, fluorescence intensity of the RNA/DNA duplexes will occasionally drop below 8,000, when the corresponding DNA/DNA duplexes will still fluoresce within the 20,000-30,000 range. In such cases, the results for RNA may be regarded as sub-optimal. A synthesis or hybridization failure, either for DNA or RNA, will be immediately noticeable during scanning from the obvious lack of fluorescence. There are multiple opportunities for the RNA synthesis to either fail or partially succeed and they will be outlined in the discussion part.
Library deprotection, cleavage and recovery 
Depending on the complexity and the density of the library, the shape and outline of the synthesized array can be seen without magnification but under proper lighting (Figure 4), with the DNA still in protected form. After deprotection with EDA/toluene, and before the addition of water in order to collect the cleaved library, the synthesis area containing the now deprotected oligonucleotides may stand out as a hydrophilic zone, when the rest of the glass slide will appear covered with a turbid hydrophobic layer. The direct observation of the synthesis area depends on the total area used to synthesize oligonucleotides: greater use of the synthesis area will correspond to a greater chance of clear distinction between hydrophilic and hydrophobic regions on the surface. Conversely, libraries synthesized using fewer mirrors and with smaller features may not be immediately observable.
Similarly, the amount of recovered DNA after desalting is directly proportional to the total area used for synthesis. If all features are used for oligonucleotide synthesis, the cleavage and recovery procedure should yield between 25 and 30 pmol of DNA. A 10% use of the synthesis area will therefore afford only around 3 pmol of DNA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59936/59936fig1v2.jpg" /> 
Figure 1. Chemical structures of the DNA and RNA phosphoramidites used in oligonucleotide synthesis by microarray photolithography. Standard nitrophenylpropyloxycarbonyl (NPPOC) photosensitive protecting groups at the 5&#39;-OH are used in regular DNA and RNA microarray synthesis for hybridization purposes. For the synthesis of complex DNA libraries, the more photolabile benzyl-NPPOC (BzNPPOC) are preferred at the 5&#39;-OH, as BzNPPOC is removed twice as fast as NPPOC, which significantly reduces total microarray synthesis time. DNA oligonucleotides for libraries also require the coupling of a cleavable dT monomer at the 3&#39;&#160;end. This monomer, which carries a succinyl ester function, will be cleaved during deprotection, allowing for the DNA to be collected from the microchip. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59936/59936fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59936/59936fig2v2.jpg" /> 
Figure 2. Example of a mask as an image file sent to the micromirror device during UV exposure. The white pixels correspond to mirrors which will be tilted in the &#34;ON&#34;&#160;position, reflecting UV light onto the synthesis cell. Black pixels correspond to &#34;OFF&#34;&#160;mirrors, where the UV light will be reflected away from the cell. White pixels will therefore allow for the coupling of the next incoming phosphoramidite on the oligonucleotides found at the corresponding features on the glass substrates. Oligonucleotides synthesized on the features whose corresponding mirrors are, in this mask file, black pixels will however remain inert during the next coupling event. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59936/59936fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59936/59936fig3v2.jpg" /> 
Figure 3. Photographs of the microarray photolithography optical and synthesis setup. (A) Optical circuit for UV exposure. UV light from the UV-LED is first homogenized through a rectangular-cross-section light-pipe then reflects onto the micromirrors. Micromirrors which have been tilted into an &#34;OFF&#34;&#160;position will reflect UV-light away from the synthesis cell, but micromirrors in the &#34;ON&#34;&#160;position will reflect light onto the synthesis cell, situated at the focal plane, by first passing through a 1:1 Offner Relay imaging system. (B) The synthesis cell, once assembled, consists of a drilled slide placed first onto the quartz block of the cell, separated by a thick PTFE gasket (not shown). A second, non-drilled slide is then positioned over the drilled slide, separated by a thin PTFE gasket. A metal frame (not shown) holds the assembly together. (C). For library preparation, once the synthesis cell is attached at the focal plane of incoming UV light, the chamber located between the quartz block and the drilled slide is filled with a 1% solution of &#946;-carotene in CH2Cl2. To do so, an additional inlet and outlet tubing is attached to the quartz block and the orange solution flows from the rightmost to the leftmost position. The flow of reagents and solvents for the synthesis is shown in white arrows. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59936/59936fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59936/59936fig4.jpg" /> 
Figure 4. The synthesis area is usually visible to the naked eye. Here, a DNA library can be seen on the glass surface right after synthesis, with the DNA still in protected form. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59936/59936fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59936/59936fig5v2.jpg" /> 
Figure 5. Hybridization assays to the 25mer DNA and RNA sequences synthesized in situ on microarrays. (A) Fluorescence scan of the entire hybridized DNA and RNA microarray. 25mer DNA and RNA oligonucleotides are hybridized to their Cy3-labelled complementary strands. The array was scanned with a laser at a 532 nm excitation wavelength, at 5 &#181;m resolution. (B) Fluorescence intensities (arbitrary units) of the DNA:DNA and RNA:DNA duplexes in three separate experiments. The light green data for in situ synthesized RNA oligonucleotides can be considered suboptimal, when compared to the fluorescence intensity of the corresponding DNA sequences. Error bars are SD. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59936/59936fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59936/59936fig6v2.jpg" /> 
Figure 6. Schematic representation of the deprotection, cleavage and recovery procedure for DNA libraries synthesized on microarrays. DNA sequences are grown on a base-sensitive cleavable dT nucleoside (shown in the zoomed area). After synthesis, deprotection of the DNA oligonucleotides (base protecting groups are represented as red spheres) in EDA/toluene leaves the deprotected material electrostatically bound to the surface and can then be pipetted out by applying a small amount of water onto the synthesized area. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59936/59936fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59936/59936fig7v3.jpg" /> 
Figure 7. Representative absorbance spectrum (220 -&#160;350 nm) of a cleaved, desalted DNA library containing 4,000 different sequences, 100-nt in length. A total of 940 ng of DNA was isolated from a single array synthesis, corresponding to 30 pmol of DNA total, or 15 pmol per glass substrate. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59936/59936fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/59936/59936table1.jpg" /> 
Table 1. Representative cycle protocol for coupling/oxidation/photodeprotection of 5&#39;-BzNPPOC-dA, assuming the corresponding phosphoramidite was loaded on port &#34;A&#34;. Coupling time (in seconds) is shown at the &#34;couple monomer&#34;&#160;line. The UV photodeprotection time, here corresponding to a radiant energy of 3 J/cm2 (BzNPPOC photochemistry), is calculated as the time elapsed between the two &#34;Event 2 Out&#34;&#160;communication signals.
<img alt="Table 2" src="/files/ftp_upload/59936/59936table2.jpg" /> 
Table 2. Representative cycle protocol for coupling/oxidation/photodeprotection of 5&#39;-NPPOC-rA, assuming the corresponding RNA phosphoramidite was loaded on port &#34;A&#34;. Coupling time (in seconds) is shown at the &#34;couple monomer&#34;&#160;line. The UV photodeprotection time, here corresponding to a radiant energy of 6 J/cm2 (NPPOC photochemistry), is calculated as the time elapsed between the two &#34;Event 2 Out&#34;&#160;communication signals.

Watch this Scientific Journal Video about High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries at JoVE.com

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

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Studying DNA and RNA

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17.9K Views.  University of Vienna. In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Video: High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as &#945;-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Summary

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Chapters in this video

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay