Reverse transcription and DNA amplification experiments with activated heat-stable DNA polymerase
Reverse transcription and DNA amplification experiments with activated heat-stable DNA polymerase
This experiment introduces activated heat-stable DNA polymerase for reverse transcription and DNA amplification. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Reverse transcription and DNA amplification experiments with activated heat-stable DNA polymerase
Materials and Instruments
RNA oligonucleotide primers Deoxyribonucleoside triphosphate RT RNA PCR enzyme AccuRT buffer UNG agarose Ethidium bromide Dimethyl sulfoxide Magnesium acetate Water to remove RNA enzyme SYBRGreen I Dye Move I. Materials For more product details, please visit Aladdin Scientific website.
Electrophoresis equipment for agarose gels
1. Buffers, solutions and reagents
Agarose
Dimethyl sulfoxide (see Note 1) (DMSO)
Ethidium bromide
Magnesium acetate (see Note 2) [( CH3COO)2 Mg] 25 mmol/L (AppliedBiosystems4339582/4339585)
Water for RNAase removal
SYBRGreen I Dye (see Note 1) (molecular probe)
2. Enzyme and enzyme buffer
5X AccuRT Buffer (see Note 2) (Applied Biosystems 4339582/4339585)
Gene Amp Accu RT RNA PCR Enzyme (see Note 2): 3.75U/ul (Applied Biosystems 4339585)
AmpErase uracil-N-glycosylase (UNG):1U/ul(AppliedBiosystems)
3. Nucleic acids and oligonucleotides
Deoxyribonucleoside triphosphate: neutral 10 mmol/L solution (AppliedBiosystems, N808-0007/N808-0095 contains 20 mmol/LdUTP in place of dTTP).
Oligonucleotide primer: 15 umol/L in 10 mmol/L Tris-HCl solution, pH 8.3.
RNA：通常保存于含载体 RNA 的溶液中，如 30ug/mlE.colirRNA 或 poly(rA)，或保存于 10 mmol/LTris-HCl、pH7.0，lmmol/LEDTA,0.lmmol/LNaCl 中，两者都需去除 RNA 酶的水。
4. Special equipment
Electrophoresis equipment for agarose gels
GeneAmpPCR System 9700/2700 (AppliedBiosystems) or equivalent real-time quantitative PCR detection system (see Note 1): e.g., AppliedBiosystems GeneAmp5700 Sequencing System or ABIPRISM7000, 7700/7900 HT Sequencing System or RocheAppliedScienceLightCycler. RocheAppliedScienceLightCycler
5. Other
MicroAmp Reactor Tubes (AppliedBiosystems) or equivalent 
Methods
1. RNA Isolation
The preparation of high quality RNA is of paramount importance and for this there are many commercially available products as well as detailed methods.
(DieffenbachandDveksler1995;Farrell1998;SambrookandRussell2001; Chapter 10).
2. Combination of RT and PCR amplification
(1) Reactions with dTTP but without UNG
(1) Thaw all reagents and shake slightly to avoid foaming.
② Dump the enzyme and reagents to the bottom of the tube in a microcentrifuge and place on ice.
③ Mix the following reagents (note: items in bold are at different concentrations in the presence of dUTP and UNG). 
④ Lightly shake the above mixture, add 1.00ul of RNA (to 1ug, see 16.1 Troubleshooting and Tips), and lightly shake the entire reaction mixture. Any ratio of RNAase-removing water to RNA may be used, as long as the total volume of the primary mixture and RNA equals 50ul.
⑤ Perform the reaction on the GeneAmpPCRSystem9700/2700 according to the following procedure.
65°C, hold for 30 min (reverse transcription)
95°C, hold for lmin
95°C, 15s; 65°C, 30s, 40 cycles (PCR amplification)
65°C, hold for 7 min
(vi) Reactants can be used directly for analysis or stored at -20°C.
(2) Reactions with dUTP and UNG (for contamination control systems and UNG-mediated hot starts)
(i) Thaw all reagents, shaking slightly to avoid foaming.
② Centrifuge the enzyme and reagents to the bottom of the tube in a microcentrifuge on ice.
(iii) Mix the following reagents (note: items in bold are used at different concentrations for reactions with and without dTTP and UNG). 
(iii) Mix the following reagents (note: different concentrations are used for reactions with and without dTTP for items in bold.) (iv) Shake the above mixture gently, add 1.00ul of RNA (to 1ul; see 16.1 QUESTIONING AND TIPS), and shake the entire reaction mixture gently. Any ratio of RNAase-removing water to RNA may be used, as long as the total volume of the primary mixture and RNA equals 50ul.
⑤ Perform the reaction on the GeneAmpPCRSystem9700/2700 according to the following procedure.
50°C, hold for 2 min (UNG step)
65°C, hold for 30 min (reverse transcription step)
95°C, lmin
95°C, 15s; 65°C, 30s, 40 cycles (PCR amplification)
65°C, hold for 7 min
(vi) Reactants can be used directly for analysis. To inactivate UNG, store at -20°C: or chemically treat (e.g., extract with an equal volume of chloroform), as prolonged presence of UNG will destroy the amplification product. Reactants can be stored for long periods at -20°C: UNG does not need to be inactivated prior to analysis, however, at room temperature or below 55°C it can destroy the replicon in the reaction.
3. Reaction Product Analysis
For detection of amplification products, electrophoresis can be performed on 2% agarose containing 0.5ug/ml ethidium bromide (EB) or by TaqMan for real-time quantitative PCR (Mengetal. 2001). Staining with EB (Higuchi and Waston 1999; Kang and Holland 1999; Kangetal.2000; Holland 2002) or SYBRGreenI (Baranzinietal.2000) was detected by fluorescence of the double-stranded DNA of the EB- or SYBRGreenI-conjugated reaction products. The detection is done by the fluorescence emitted by EB or SYBRGreenI binding to the reaction product, double-stranded DNA. Concentration control is important when using SYBRGreenI, with a 20-fold master mix in 100% DMSO (20-fold master mix refers to 500-fold SYBRGreenI in DMSO solution), and a final concentration of 0.2-fold being used, above which it has been shown to inhibit many of the enzymes of the RT reaction.