FAQs for Reverse Transcriptase PCR (RT PCR)

IS IT NECESSARY TO DIGEST FIRST-STRAND cDNA MADE WITH SUPERSCRIPT II REVERSE TRANSCRIPTASE (RT) WITH RNase H? It is not always necessary. For many primers, PCR products are seen without theRNase H treatment. Since SUPERSCRIPT II RT lacks RNase H, the unnicked RNA/cDNAhybrids may not denature well during the initial denaturation in PCR.  Sometemplates and primers may require the RNase H digestion. For some RNA template/primer sets, the RNase H step may be helpful even when the cDNA is made with an RNase H containing reverse transcriptase such as mMLV RT.IS IT NECESSARY TO DIGEST FIRST-STRAND cDNA MADE WITH THERMOSCRIPT REVERSE TRANSCRIPTASE WITH RNase H?It is not always necessary. For many primers, PCR products are seen without the RNase H treatment.  Since THERMOSCRIPT RT essentially is RNase H minus, the unnicked RNA/cDNA hybrids may not denature well during the initial denaturation inPCR and therefore may not yield a subsequent PCR product.  If a PCR product is notobtained when an RNase H step is not included after cDNA synthesis, always repeat thePCR after an RNase H treatment.FOR FIRST-STRAND SYNTHESIS OF cDNA FOR RT-PCR, IS IT BETTER TO PRIME WITH OLIGO(dT), RAMDOM HEXAMERS, A GENE-SPECIFIC PRIMER, OR A COMBINATION OF THESE PRIMERS?It depends on your experimental goals. Oligo(dT) is used for many systems and is keyfor full-length cDNA. Random hexamers give a series of short first-strand productsspanning the entire mRNA. Use of random hexamers may be helpful if your PCR fragmentis at the 5 end of a large mRNA. To ensure representation of the 3 end, oligo(dT)is added with random hexamers. Gene specific primers for cDNA synthesis are used ina few applications such as 5 RACE. GSP may not work in cDNA synthesis even if itdoes work in PCR so it's best to try a couple of your having problems.HOW CAN I TRAAT MY RNA SAMPLE BEFORE RT-PCR TO ELIMINATE DNA CONTAMINATION?  Combine 1 g total RNA, 1 l 10X DNAse I buffer (200 mM Tris-HCl (pH 8.4),500 mM KCl, 20 mM MgCl2), 1 l DNAse I, Amplification Grade, 1 unit/l and DEPC treated water to 10 l. Incubate for 15 min at room temperature. Inactivate by adding 1 l of 25 mM EDTA and heat for 10 min at 65 C. Note: 1 unit of DNAse I should be enough to treat up to ~10 g of RNA.WHAT IS THE HIGHEST TEMPERATURE AT WHICH SUPERSCRIPT II CAN BE USED?For RT-PCR, SUPERSCRIPT II RT can be used up to 55 C. Be sure your first-strand primer anneals at the high temperature. WHAT IS THE HIGHEST TEMPERATURE AT WHICH M-MLV REVERSE TRANSCRIPTASECAN BE USED?It can be used at temperatures up to 42oC. WHAT IS THE HIGHEST TEMPERATURE AT WHICH THERMOSCRIPT REVERSE TRANSCRIPTASE CAN BE USED?It can be used at temperatures as high as 70oC (especially for amplicons expectedto be 1 kb or less).  For PCR products expected to be greater than 1 kb a maximumfirst strand synthesis temperature of 65oC is suggested.IN A TWO-STEP RT-PCR, HOW MUCH OF THE FIRST-STRAND cDNA REACTION IS USED FOR PCR?Use 10% of the first-strand reaction. More than 10% may inhibit the PCR.HOW CAN I TELL IF MY RT-PCR PRODUCT IS RNA SPECIFIC?Include a control reaction without reverse transcriptase. Alternatively, a primer set that spans an intron/exon border can be chosen such that the RNA dependent PCR product would be a different size from genomic DNA-dependent PCR product.HOW DOES THE SUPERSCRIPT PREAMPLIFICATION SYSTEM DIFFER FROM THE SUPERSCRIPT ONESTEP SYSTEM FOR RT PCR?                  SuperScript One Step       SuperScript Preamplification SystemNumber of Steps           One                             TwoPrimer Choice        Gene Specific         Gene Specific, Oligo d(T), Random PrimersSensitivity         Down to 5-10 Copies    10-1000 less sensitive than One StepOptimization   Required         Not Really                        YesHOW DOES THE THERMOSCRIPT RT-PCR SYSTEM COMPARE WITH THE SUPERSCRIPT ONESTEP AND SUPERSCRIPT PREAMPLIFICATION SYSTEMS FOR RT-PCR?The THERMOSCRIPT system uses THERMOSCRIPT Reverse Transcriptase which is an avianreverse transcriptase that is essentially RNase H minus.  It is a morethermostable enzyme.  This is a two step system that allows the user to choose oligo d(T), random primers or gene specific primers for first strand synthesis.

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