This procedure begins with sectioning eight micrometer liquid nitrogen vapor frozen tissue blocks on a cryostat set at minus 17 degrees Celsius onto plain uncharged glass slides. Thereafter, the sections are stained with the histo gene staining kit. After identifying the pyramidal neurons, the cells about 500 are laser captured onto the HS capture cap.
The cap with cells is placed in a micro centrifuge tube containing 50 microliters of extraction buffer turned upside down and placed in a Falcon tube previously positioned in Iraq, seated in a water bath set to 42 degrees Celsius. After a short centrifugation step, the cap is removed. The remaining solution is then ready for RNA isolation.
Hi, I'm Charmaine Peterson from the laboratory of Wilson Wu in the Department of Structural and Molecular Neuroscience at McLean Hospital. Today we're going to show you a procedure for laser capturing parametal neurons from human postmortem brain tissue. We use this procedure in our laboratory to study differential gene expression in the superiors and poor gyrus of schizophrenia subjects using microarray technology.
So let's get started. Brain tissues were obtained from the Harvard Brain Tissue Resource Center as liquid nitrogen vapor frozen blocks prior to tissue sectioning. It is important to reduce RNAs contamination all surfaces, including the work area, sectioning blade and slides are treated with an RNA decontamination solution, such as RNA SAP and wiped down with 100%ethanol.
This procedure begins with sectioning liquid nitrogen vapor frozen tissue blocks on a cryostat set at minus 17 degrees Celsius onto plain uncharged glass slides. The tissue sections are now ready for the identification of pyramidal neurons using the histo gene quick staining kit. Prepare the ethanol dehydration series aliquot 25 milliliters of the appropriate ethanol concentrations and RNA free water into the staining jars provided with the histo gene kit.
Place all jars in an ice bucket except for one 75%ethanol jar. Place that 75%ethanol in the minus 20 degrees Celsius freezer. Next, prepare the staining solution by adding one microliter of RNAs inhibitor per 100 microliter of staining solution.
Since we will be staining four tissue sections on two slides, four microliters of RNAs inhibitor is added to 400 microliters of the staining solution in a micro centrifuge tube. Keep the staining solution on ice. Under a fume hood.
Add molecular sieves into the xylene jar to remove excess water, which could compromise tissue lift. Switch on the water bath with the temperature set to 42 degrees Celsius and place a 50 milliliter Falcon tube supported by a rack in the water bath. Remove two slides from the minus 80 degrees Celsius freezer and defrost them on a Kim wipe.
For approximately 30 seconds or just until the corners of the slides start to defrost, briefly fix the tissue in the 75%ethanol for 30 seconds in the minus 20 degrees Celsius freezer. Then using RNA free forceps, transfer the slides to nuclease free water for a 32nd wash After the slides have been washed, outline the sections with a PAP barrier pen to concentrate the staining solution on the section stain the four sections with the histo gene staining solution for 20 seconds. With 97 microliters of the stain slash RNAs inhibitor per section dehydrate the sections in the previously prepared ethanol on ice for 30 seconds per step.
The final 100%ethanol step should be extended to three minutes to achieve sufficient dehydration for adequate tissue lift. Finally, immerse the slides in xylene for five minutes, allowing the slides to air dry completely before proceeding to laser capture microdissection, the pyramidal neurons must be immediately removed after staining the procedure for single cell laser capture. MICRODISSECTION or LCM requires a 42 degrees Celsius water bath containing a 50 milliliter Falcon tube supported by a, which was set up earlier.
Single cell LCM is accomplished with the ARCTURUS XT laser capturing system and software. To begin this procedure, load the slides and caps onto the arcturus XT apparatus. Use the capture HS caps, but keep the program setting on macro.
Click on the box load with overview to obtain an overview photo of each slide. Adjust the brightness focus at two x magnification to determine the optimal section for laser capture. Avoid tissue sections with excessive folding, but choose sections that are intact, smooth, and stained.
Well place a cap over the general area where you will be capturing, making sure to include the area to be captured in our case. Layer three of the cortex. Make sure that the cap rails do not rest on any folds as this will tilt the cap resulting in variable spot sizes.
Next, confirm the location of the IR laser spot manually at the 40 x magnification. The blue cross should be centered within the IR laser spot. If not, adjust its location by right clicking on the spot and selecting located IR spot.
At 40 x magnification, identify pyramidal neurons according to the following criteria, one that cells our pyramidal in shape and two, the proximal portion of the apical and or basal dendrites are identifiable. Save the position of the cap by clicking on the plus sign at the position function. This way, if the cap is moved, it will always return to precisely the same spot for which you'd adjusted the spot size.
Enter these values into the control box 70 into power and 16 into duration. As these parameters are specific for our tissue, we recommend that you should test and adjust to these variables according to your specific tissue sample before commencing. With the laser capturing of single cells, unclick the auto move stage option and make sure that the right size symbol correlates with the symbol on the panel to the right.
Select the circle option in the bottom right to select a neuron that you would like to test capture. And after aligning the blue cross with the circle, activate the laser by clicking on test IR spot. The spot made by the laser should firstly have a crisp dark ring around the object captured.
Make sure that the ring is big enough to encompass the cell, but small enough that it doesn't include unwanted tissue or other cells. If this ring is too light, the cell was not captured. If there is a dark spot in the middle of the dark ring, then the laser strength slash duration is too great.
Repeat this process on different parts of the tissue within the layer that you wish to capture. To check that the spot size does not differ depending on the location, adjust accordingly. Identify pyramidal neurons for capture approximately 500 cells.
Press the laser capture button to capture cells. Move the cap to the QC station. Make sure that at least 90%of the neurons were removed.
If not, capture more cells in the area where most of the cells were captured. Place the cap into a 0.5 milliliter micro centrifuge tube containing 50 microliters of extraction buffer. The cap has been designed to fit perfectly to prevent the buffer from leaking.
Turn the assembly upside down, making sure that the extraction buffer covers the entire cap and place it at the bottom of the 50 milliliter Falcon tube in the water bath set at 42 degrees Celsius. Incubate the neurons for 30 minutes to remove the tissue from the cap after the incubation centrifuge the tube and cap assembly for two minutes at 800 gs. After the centrifugation, remove the cap.
If RNA isolation will only be performed at a later time. Store the remaining cell extract at minus 80 degrees Celsius. Otherwise proceed with isolating RNA from the cell extract as shown in the next section, RNA isolation is performed using the PICO pure isolation kit, which is designed to isolate small numbers of cells from LCM samples and to retain low abundance mRNA.
To begin this procedure, add 70%ethanol provided with the kit to the cell extract and centrifuge on a preconditioned purification column. In order to bind the RNA to the column filter after washing, treat the RNA with D Ns to eliminate the risk of DNA interference. This step is particularly important in downstream applications such as real-time R-T-P-C-R.
After the DNA digestion add 40 microliters of wash buffer one and centrifuge the purification column for 15 seconds at 8, 000 Gs.Thereafter, proceed with the washes according to the protocol provided with the kit, but extend final wash step with wash buffer two from two to two and a half minutes to ensure that no wash buffer remains in the column, which can reduce your RNA yield. Transfer the column to another micro centrifuge tube. Add the elucian buffer and incubate on the filter in the column for one minute centrifuge the column to elute the RNA.
Finally, check the quality of the extracted RNA by running an Experian High Sense lab chip, which provides a virtual gel and electropherogram pipette 1.3 microliters of the sample into a 0.5 milliliter tube. For the quality control test, freeze the rest of the sample at minus 80 degrees Celsius. Once the quality of the RNA is verified, it can be amplified by and labeled and hybridized.
For gene expression Profiling analysis, two rounds of linear amplification will be performed with the ribo amp HS plus kit, which should result in approximately 50 micrograms of amplified RNA sufficient for the performance of both microarray and Q-R-T-P-C-R experiments. The turbo biotin labeling kit and labeling from molecular devices is used to label the amplified RNA. Finally, gene expression profiling using the human X three P gene chip pro beret from atrix will be performed.
Tissue sectioning should result in two slides with two sections per slide, four sections in total per case. Each section should be smooth with minimal tearing, cracking, or folding. The pyramidal neurons should be darkly stained around 20 to 25 micrometers in size with a pyramidal shape and visible apical dendrites.
During LCM, after the laser has pulsed through the thermoplastic film, the cells adhere to the cap and are therefore no longer on the slide leaving surrounding tissue. Behind approximately 85 to 100%of the neurons should adhere to the cap with the capture HS caps at macro settings and correct adjustment of the laser power and strength. For each section, you should obtain around 500 to 700 cells per section resulting in at least 500 picograms of total RNA per case.
After total RNA isolation, the RNA quality is evaluated by means of an electropherogram and a virtual gel via the BioRad Experian. In the electropherogram, you should see two distinct peaks corresponding to the 18 s and 20 a s ribosomal RNA units with postmortem tissue. However, this is not always the case as the tissue may be degraded due to factors prior to sectioning.
You will normally see a large bump indicating degradation with a large peak around 18 s and a smaller 28 s peak as indicated by the red arrows. In contrast, bad quality RNA with too much degradation will be indicated by an electropherogram with a large area under its curve. In addition to a downshift in the location of the spread to test the quality of the mRNA after two rounds of linear amplification, we use both the BioRad Experian standard Sense lab chip and the NanoDrop spectrophotometer.
Traditional Atrics microarray technology requires the mRNA transcript spread to be at least 600 nucleotides in length in order to be detected with this protocol. The mRNA spread reached the 1000 nucleotide range as indicated by the electropherogram with large peaks slowly descending as a function of time. This result is also confirmed by the virtual gel.
If the transcript lengths are shorter than 600 nucleotides, the sample should not be included For hybridization, the NanoDrop readings indicated an average a two 60 over a two 80 ratio or purity of 2.5 across samples. The average concentration was 1.7 micrograms per microliter, resulting in approximately 50 micrograms of mRNA per sample, sufficient for both microarray analysis and subsequent validation, which requires between 15 to 20 micrograms of mRNA and subsequent validation of the results with QR TPCR. Our results indicate that with this protocol, both the quantity and quality of RNA obtained is good enough to investigate gene expression differences via the atrix human X three P chip.
After hybridization to the Atrics Human X three P chip, we achieved percent calls of an average of 26.6%indicating adequate hybridization and probe intensities. We have just shown you how to laser capture parametal neurons from postmortem human brain tissue in order to use the RNA obtained from these cells. For microarray G profiling studies when doing this procedure, it is important to perform all steps in an RNAs free environment and to complete the steps in a timely manner in order to preserve RNA integrity.
So that's it. Thanks for watching and good luck with your experiments.
