RT-PCR

Background: Reverse transcriptase polymerase chain reaction (RT-PCR) is a method used to amplify cDNA copies of mRNA. It is an extremely useful technique in RNA semi-quantitation, cloning, and probe synthesis. The technique consists of two main steps. In the first step (first strand reaction), complementary DNA (cDNA) is made from an mRNA template using dNTPs and reverse transcriptase enzyme. In the lab we use Superscript II First Strand Synthesis kit from Invitrogen. After the reverse transcriptase reaction is complete and cDNA has been generated from the mRNA, standard PCR (second strand reaction) is performed with Taq DNA polymerase and gene-specific primers. Before beginning: (1) Set up reverse transcriptase (RT) reactions at the bench labeled RNA ONLY. (2) Make sure to use a new set of gloves while working with RNA so that you avoid RNAse activity. RNAses degrade RNA and are found everywhere, especially on your hands. Before starting use RNAse ZAP (from Ambion) which is located on the RNA ONLY bench to wipe down the bench, pipettes, and your hands. RNAse ZAP removes RNAse contamination from glass and plastic surfaces. Also, use DEPC H20. DEPC water contains Diethyl Pyrocarbonate, which degrades any trace of RNAses. (3) When setting up the RT reaction use filtered RNAse free tips, which can be found along with the proper tubes for RT in the drawer labeled RNA supplies (located under the bench labeled RNA ONLY). (4) In the lab we often use the Superscript II First Strand synthesis kit from Invitrogen (catalog # 11904-018). If any of the components run low you can purchase another kit from the stockroom. You will need buffer, oligo d(T) or random oligomer, dNTPs, DEPC water, Superscript II reverse transcriptase, MgCl2, DTT, RNAse Out, and RNAse H. They can be found in the yellow stratacooler labeled RT-PCR in the -20oC freezer. If any

reagents run low there are extra vials located on the lowest shelf on the door of the -20oC freezer in a box labeled RT-PCR accessories. An alterative is the iScript kit from BIORAD, which involves a much more simpler protocol. (5) The entire procedure may be done in the icycler PCR machine (there is a protocol saved on the machine) or you can use water baths/heatblocks to incubate the samples for the various incubation steps. Setting up Reverse Transcriptase Reaction: (1) In the first step of the reaction, we typically use 2 mg of total RNA. The source of RNA can be from tissues or from cultured cells. Oligo dT is the primer of choice. However, if you need to isolate 5end of the cDNAs or longer cDNAs you can use a random oligomer to facilitate the reverse transcriptase enzyme to proceed all the way to the 5end of the mRNA. The reaction is incubated at 65oC for 5 min to allow Oligo dT to anneal to the mRNA. Note: The quality of RNA is very important, if needed poly (A) RNA can be used. It is useful to check the RNA quality by running it on a gel. (2) In the next step of the reaction, additional reagents are added. The 10 buffer contains Tris, KCl. MgCl2 is added to the reaction in order to stabilize ATP and aid in the transfer of the phosphate group during extension. Also, DTT (dithiothreitol) is added, which is a reducing agent that breaks disulfide bonds. Lastly, add RNase OuT, which is a potent non-competitive inhibitor of ribonucleases such as RNase A, B, and C, but not H. The reaction is incubated at 42oC for 2 min to allow the reaction to get to optimal temperature for reverse transcriptase. (3) Next, Superscript II Reverse Transcriptase is added and the reaction is incubated at 42oC to allow extension. This is the step where cDNA is synthesized from the RNA template. (4) The reaction is then terminated at 70oC. RNase H is added. RNase H is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA in RNA:DNA duplexes to generate products with 3 hydroxyl and 5 phosphate ends. It will not degrade single-stranded or double-stranded DNA or RNA.

(5) There are three types of DNA primers commonly used to facilitate reverse transcriptase binding and function: random primers, oligo (dT) primers and gene specific primers. Oligo (dT) primers consist of short strands (12-18 bases) of thymine deoxynucleotides that hybridize to the mRNA poly A tail and prime reverse transcription. These maximize the presence of long cDNAs with many mRNA 3 ends in the final cDNA population. (6) Wherever possible, oligonucleotide primers that bind to sequences located in different exons of the target RNA should be used as sense and antisense primers for amplification of the cDNA product. In this way, amplification products derived from cDNA and contaminating genomic DNA can be easily distinguished. (7) Once, the cDNAs are generated, proceed with PCR for housekeeping gene (Actin, GAPDH) and then the gene of interest. Example: This is the protocol that is used when using the Invitrogen Superscript II First Strand Synthesis kit (Please note that other kits use different amounts of reagents) 1. Set up reaction on the RNA ONLY bench after wiping it off with ZAP. Make sure to use filtered RNA ONLY tips. Use RNase free tubes, if you want to use the PCR machine, use appropriate tubes. Sample 1 +RT 2ug RNA (1ug/ul) 10mM dNTPS Oligo d(T) (0.5ug/ul) DEPC H20 to 10ul Total Sample 1 RT 2ul 1ul 1ul 6ul 10ul Master Mix (3) 6ul 3ul 3ul 18ul 30ul (8ul/sample)

2ul 1ul (Final concentration: 0.5mM) 1ul 6ul 10ul

2. Incubate at 65oC for 5 min to denature RNA secondary structure 3. Put on ice for 1 min 4. Set up reaction: Sample 1 +RT 10x buffer 2ul (Final concentration: 1) Sample 1 RT Master Mix (x3) 2ul 6ul

25mM MgCl2 0.1M DTT RNase out (add last) (40units/ul) Total Total volume

4ul (Final concentration: 5mM) 2ul (Final concentration: 0.01M) 1ul 9ul (add to sample) 19ul

4ul 2ul 1ul 9ul 19ul

12ul 6ul 3ul 27ul (9ul/sample)

5. Incubate at 42oC for 2 min (optimal temperature for Superscript II reverse transcriptase). However, can incubate up to 50oC. 6. Add 1 ul Superscript II Reverse Transcriptase (RT) (50units/ul) to each sample except to Sample 1 -RT. You may use a different reverse transcriptase like Thermoscript but you want the reverse transcriptase with reduced RNase H activity because this results in greater full-length cDNA synthesis. 7. Incubate at 42oC for 50 minutes as this is the optimal temperature for Superscript II reverse transcriptase. Use a different temperature as recommended for other reverse transcriptases. 8. Terminate reaction at 70oC for 15 min and then cool on ice (4oC). 9. Add 1ul RNase H (2units/ul) to each tube and incubate at 37oC for 20 min because RNase H degrades mRNA and at this point you only want cDNA not mRNA. Store products at -20oC for long term usage.