1. Quant Reverse Transcriptase For first-strand cdna synthesis and two-step RT-PCR www.tiangen.com
RT080530 Kit Contents Quant Reverse Transcriptase Contents Cat. no. ER103 ER103-02 25 rxns ER103-03 50 rxns ER103-04 100 rxns Quant Reverse Transcriptase 25 μl 50 μl 2 50 μl 10 RT Buffer 70 μl 150 μl 2 150 μl Handbook 1 1 1 Storage Quant Reverse Transcriptase should be shipped on dry ice and stored immediately upon receipt at 20 C. Introduction Quant Reverse Transcriptase is a new, unique enzyme, different from the reverse transcriptases of Moloney Murine Leukemia Virus (MMLV) or Avian Myeloblastosis Virus (AMV). Quant Reverse Transcriptase is a recombinant heterodimeric enzyme expressed in E. coli. It has a high affinity for RNA, which enables efficient and sensitive reverse transcription of RNA templates with high GC content or complex secondary structures, leading to high yields of cdna. Application RT-PCR, Real-Time PCR, 3 and 5 RACE PCR, prime extension, cdna library construction, serial analysis of gene expression (SAGE), etc. Quant Reverse Transcriptase Handbook 1
Important Notes 1. The protocol is optimized for use with 50 ng to 2 μg of RNA. With >2 μg RNA, scale up the reaction linearly to the appropriate volume. 2. Set up all reactions on ice to minimize the risk of RNA degradation. 3. Separate denaturation and annealing steps are generally not necessary. However, for some RNAs with a high degree of secondary structure, a denaturation step may be desired. If so, denature the RNA in RNase-free water before reaction setup: incubate the RNA for 5 min at 65 C, then place immediately on ice. Do not denature the RNA in the reaction mix. 4. When using oligo-dt primers, a primer length of at least 12 nucleotides and a final concentration of 1 μm is recommended. 5. Operate carefully to minimize RNA degradation and cross contamination. Starting Template Reverse transcriptases are used in vitro for first-strand cdna synthesis with RNA as the starting template. The efficiency of the reaction is highly dependent on the quality and quantity of the starting RNA template. 1. It is important to have intact RNA as starting template. Even trace amounts of contaminating RNases in the RNA sample can cause RNA cleavage, resulting in shortened cdna products. 2. Chemical impurities, such as protein, poly-anions (e.g., heparin), salts, EDTA, ethanol, phenol, and other solvents, can affect the activity and processivity of the reverse ranscriptase. Quant Reverse Transcriptase Handbook 2
Protocol The protocol is optimized for use with 50 ng to 2 μg of RNA. With >2 μg RNA, scale up the reaction linearly to the appropriate volume. 1. Thaw template RNA on ice. Thaw the primer solutions, 10x RT Buffer (supplied), Super pure dntp, and RNase-free ddh 2 O at room temperature (15 25 C). Place on ice immediately after thawing. Mix each solution by vortexing, and centrifuge briefly to collect residual liquid from the sides of the tubes. 2. Dilute the RNA inhibitor to final concentration of 10 U/μl in 1x RT Buffer which has been pre-cooled in ice. Mix thoroughly and carefully by vortexing for no more than 5 sec. Centrifuge briefly. 3. Prepare a fresh master mix on ice according to Table 1. Mix thoroughly and carefully by vortexing for no more than 5 sec. Centrifuge briefly and then store on ice. Add the RNA template in the step 5. Note: 10 μl reverse-transcription reaction volume could be set up for downstream qpcr. All the components can be reduced to 1/2 to adapt for SuperReal PreMix (SYBR Green) (FP204-01, 50 μl 50 rxns; FP204-02, 50 μl 200 rxns). 4. If setting up multiple reverse-transcription reactions, distribute the appropriate volume of master mix into individual reaction tubes. Keep tubes on ice. 5. Add the template RNA (50 ng-2 μg) to the individual tubes containing the master mix. Mix thoroughly and carefully by vortexing for no more than 5 sec. Centrifuge briefly. 6. Incubate for 60 min at 37 C. 7. Add an aliquot of the finished reverse-transcription reaction Quant Reverse Transcriptase Handbook 3
to the PCR mix. Note: Put the reverse-transcription products on ice first and then apply them in following PCR amplification. For longterm storage, store the products at 20 C. The first strand cdna synthesis reaction mixture should not comprise more than 1/10 of the total PCR reaction volume. Table 1. Reverse-Transcription Reaction Components Component Volume/Reaction Final Concentration 10x RT Buffer 2 μl 1 Super pure dntp (2.5 mm each) 4 μl 0.5 mm each dntp Oligo-dT (10 μm)* 2 μl 1 μm RNA Inhibitor 1 μl 10 U (for 20 μl reaction system) Quant Reverse Transcriptase RNase-free ddh 2 O Template RNA added in the step 5 Total reaction volume 1 μl X μl X μl 20 μl (for 20 μl reaction system) PCR Amplification Two Step RT-PCR In Two-Step RT-PCR, an RNA strand is first reverse transcribed into its DNA complement; the cdna products can then be used for PCR amplification. RT and PCR are preformed separately in different tubes subsequently. Both random primers and gene specific primers can be used in Two-Step RT PCR, according the following Quant Reverse Transcriptase Handbook 4
steps: 1. Synthetize the first-strand cdna with RNA as the starting template according to the above procedure. 2. Add the cdna products into PCR reaction system. The first strand cdna synthesis reaction mixture should not comprise more than 1/10 of the total PCR reaction volume. 3. Perform the PCR amplification. One Step RT-PCR One step RT-PCR performs RT as well as PCR in a single buffer system. The reaction proceeds without the addition of reagents between the RT and PCR steps. First, cdna first-strand is amplified at 37 C, at which temperature the activity of Taq DNA polymerase is pretty low. When the reverse transcription is completed, increasing temperature inactivates the reverse transcriptase, and Taq DNA polymerase start to synthetize cdna. Since the RT and PCR are performed in the same tube, the buffer system cannot be optimized separately for these two reactions. Furthermore, reverse transcriptase has low activity in some PCR buffer. Therefore, One Step RT-PCR is not recommended. Quant Reverse Transcriptase Handbook 5
Ordering Information RNA Isolation Product Size Cat.no. RNAprep pure Kit (For Cell/Bacteria) 50 preps DP430 RNAprep pure Kit (For Tissue) 50 preps DP431 RNAprep pure Kit (For Plant) 50 preps DP432 Real Time PCR Product Size Cat.no. SuperReal PreMix (SYBR Green) 50 μl 50 rxns 50 μl 200 rxns FP204-01 FP204-02 Quant Reverse Transcriptase Handbook 6
PCR Product Size Cat.no. 2 Taq PCR MasterMix (with loading dye) 1 ml 5 1 ml KT201-01 KT201-02 Quant Reverse Transcriptase Handbook 7