The overall goal of the following experiment is to analyze gene expression in tissues and developmental stages of emerald ash Boer using quantitative PCR. This is achieved by dissection of larval tissues and categorization of developmental stages. As a second step extract, RNA from tissues and developmental stages using triol reagent, which is then used as a template to generate the first strand, CDNA, following the synthesis of first Strand CD NA quantitative R-T-P-C-R is performed.
Results are obtained that show differential expression of the target gene in various larval tissues and developmental stages based on full change data obtained from the cycle threshold values. Post quantitative R-T-P-C-R amplifications. Hi, I'm Prakash Mi, the PI for this lab in the Department of Entomology at the Ohio State University.
My lab mainly forces the genomic or the molecular aspects of the emal, lash, burer, and ash interactions. The emal, lash Burer or EAB is an exotic invasive pest from China and it threatens the very existence of ash populations throughout North America. Today we'll show you a protocol for learning gene expression analysis in the EAP re using QPCR or quantitative real time PCR.
And the lab members that will be involved in this protocol are Swapna pri ou, who will be doing the val dissects and then bin Bandari will be showing the RNA isolation. And finally, Lauren Rivera Vega will be looking at QPC analysis. We also use this procedure in our laboratory to study the gene expression of ash.
So let's get started. Prior to the start of this procedure, place emerald ash B or EAD larvae on moist tissue paper. Until dissections are performed, keep freshly prepared one x phosphate buffered saline or PBS on ice to keep the buffer cold throughout the dissection.
To begin, fix the EAB larvae on the dissection plate with fine dissection pins. Then add cold one XPBS to the dissection plate. First decapitate larvae with a fine pair of dissection scissors.
Subsequently, remove the last abdominal segment of the larvae using a watchmaker pair of forceps. Lift the cuticle from the larval carcass, then make an incision along the length of the carcass using a pair of scissors. Take care not to rupture the gut.
As the incision is made carefully isolate the midgut tissue from the other tissues such as fat bodies and connective tissue. Then rinse the tissue in fresh one XPBS buffer to ensure that there is no contamination of fat body. Transfer the midgut to 500 microliters of pre chilled triol reagent in a 1.5 milliliter eend orph tube.
Next, isolate the fat body tissue using forceps and transfer it into a 1.5 milliliter eend orph tube containing 500 microliters of chilled triol reagent. Finally, isolate the cuticle tissue from the larval carcass by scraping off any adhering tissue. Taking care not to damage tissue integrity.
Rinse the isolated cuticle tissue in fresh PBS buffer and transfer it to a 1.5 milliliter eend orph tube with 500 microliters of chilled triazole reagent. The isolated fat body cuticle and midgut tissues can be stored at minus 80 degrees Celsius until RNA extraction. In preparation for RNA extraction, sort the various EAB samples according to the developmental stages.
These stages include first, second, third, and fourth in stars, PrepU, and adults. To begin RNA extraction, first use plastic pestles to homogenize the tissues in the triol containing 1.5 milliliter einor tubes. After homogenization, bring the total volume of each sample to one milliliter with the triol reagent.
For the developmental stages, homogenize whole animal in liquid nitrogen with a mortar and pestle. Then add an aliquot of the homogenate to one milliliter of triol. Incubate the sample tubes with one milliliter of triol for 15 minutes at room temperature.
Following incubation, add 200 microliters of chloroform to each tube immediately after adding chloroform. Shake the tubes vigorously for about 15 seconds. Then incubate the tubes at room temperature for an additional two to three minutes.
Next, centrifuge the samples at 12, 000 GS for 15 minutes at two degrees Celsius. After centrifugation, the RNA will be in the aqueous phase. The volume of the aqueous phase should be 600 microliters or 60%of the total triol volume.
Carefully remove only the aqueous phase. Presence of substances below the aqueous phase will cause contamination of the extracted RNA. Then transfer the aqueous phase into an appropriately labeled 1.5 milliliter einor tube.
To precipitate the RNA from the aqueous phase mix, 0.5 milliliters of isopropyl alcohol to each tube. Incubate the samples at room temperature for 10 minutes. After the incubation centrifuge the tubes at 12, 000 Gs for 10 minutes at two degrees Celsius Following centrifugation, discard the S natin from the tubes.
RNA is present in the gel-like pellet formed at the bottom of the tube. Wash the pellet with 75%ethanol, adding at least one milliliter of 75%ethanol per one milliliter of mixed by vortexing. Then centrifuge the tubes at 7, 500 G for five minutes at two degrees Celsius.
Discard the supernatant from the tubes. Centrifuge the tubes again in the small table centrifuge for one minute. Remove any excess super name from the tube by careful pipetting.
Leave the tube open and let the pellet air dry for five to 10 minutes. Be careful not to over dry the pellet as this will significantly decrease its solubility after air drying. Resus suspend the pellet in dathyl percarbonate or dep sea treated water.
The amount of water used to resuspend the pellet will depend on the size of the pellet. Use 50 microliters of dep sea treated water for a small pellet or 100 microliters for a big pellet. Let the pellet completely dissolve in dep sea treated water by moving the pipette tip up and down several times.
Once the pellet has dissolved, place the sample at 55 degrees Celsius for 10 minutes. Then measure the concentration of each RNA sample using a NanoDrop spectrophotometer. Finally, perform first strand CD NA synthesis for the respective RNA samples using the in vitrogen superscript first strand synthesis system for R-T-P-C-R.
This procedure can be found in the written portion of this protocol. To begin quantitative real-time PCR or Q-R-T-P-C-R design each primer to have a melting temperature of 60 degrees Celsius and a product size of approximately 100 base pairs. AP par one is used as the gene of interest for this experiment.
A reference gene or internal control is needed for later analysis and normalization of the data. Here, ribosomal protein a P RRP one is used as the reference gene. Next, design a template indicating the sample name for each well and the QR tpcr plate.
Remember to include standards for each gene and at least two technical replicates per sample. Then turn the QRT PCR R machine on and enter the cycling parameters to set up the plate for the CFX 96 machine. First, prepare the master mix for each gene in a separate tube for each well.
The master mix consists of two microliters of nuclease free water, four microliters of 2.5 x cyber green, one microliter of primer forward and one microliter of primer. Reverse for a total volume of eight microliters according to the previously designed template, add two microliters of CDNA to each. Well then add eight microliters of the master mix to each well, resulting in a total volume of 10 microliters per reaction pipette up and down to make sure the sample is well mixed.
Check that no liquid stays on the tip. To avoid cross contamination, use a new tip for each sample. Once the entire plate is set up, cover the plate with optical tape.
Avoid touching the tape as the presence of grease can affect the reading. Then centrifuge the plate at 500 RPM for one minute to ensure that all the products in the wells are at the bottom of the plate following centrifugation. Check that there is no ice or water on the bottom of the plate.
Finally, place the plate in the QR TPCR machine and run the PCR program representative results for larval EAB. Dissection and QR TPCR are shown here. Larva EAB dissection should reveal the midgut in the middle of the larva.EAB.
The midgut of larva EAB is isolated as shown here, mean relative expression values or RAVs. For the para trophin gene AP par one are shown here for different larval tissues, including cuticle, mid gut and fat bodies. The EAB specific ribosomal protein ARP one was used as the internal control for normalizing the data obtained for the gene of interest.
The relative fold change of AP AP par one was extracted for the different larval tissues. The cuticle tissue showed the least expression and was therefore taken as the calibrator sample mean RVs for AP Perry. One in different developmental stages of EAB including the first, second, third, and fourth LAR in stars.
The PrepU and adults were determined using aprp one as the internal control to normalize the data. The relative fold change of AP Perry one was determined for various developmental stages. Here the PrepU sample showed the least level of mRNA and was therefore taken as the calibrator.
We've just shown you how to perform lateral dissections and subsequently gene expression analysis using the lateral, as well as various developmental stages of Emeral ra using quantitative R-T-P-C-R while doing this procedure. You should be careful while dissecting so that you do not cross contaminate tissue samples. And also care must be taken while performing Q-R-T-P-C-R so that you pipet the respective samples in the respective PCR blade.
So that's it. Thank you for watching our video and good luck with your experiments.
