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Products for RT-PCR, FAQ
Q 01: What is RT-PCR?
RT-PCR is a technique which couples reverse transcription (RT) of an RNA template with polymerase chain reaction (PCR) amplification of the resulting cDNA. RT-PCR is a sensitive and versatile method which can be used to determine the presence of a transcript, to estimate expression levels and to clone cDNA products without the necessity of constructing and screening a cDNA library.
Q 02: What is the RobusT™ RT-PCR Kit?
The RobusT™ RT-PCR Kit is designed for the reverse transcription (RT) and polymerase chain reaction (PCR) amplification of a specific target RNA from either total RNA or mRNA. This one tube, two-enzyme system provides sensitive, quick and reproducible analysis of even rare RNAs. The system uses Avian Myeloblastosis Virus (AMV) Reverse Transcriptase for first strand cDNA synthesis, and DyNAzyme™ EXT DNA Polymerase for second strand cDNA synthesis and DNA amplification.
Q 03: What advantages does the RobusT™ RT-PCR Kit have compared to other commonly used methods?
The RobusT RT-PCR Kit reactions are performed in a single tube without secondary additions to the reaction mix. This decreases hands-on time, relative to some commonly used methods, and minimizes the likelihood of introducing contaminants into the reaction. Furthermore, this two-enzyme system is more sensitive than a single-enzyme, rTth DNA polymerase-based system for amplification of a target in a total RNA preparation.
Q 04: Can total RNA be used as template or is poly(A)+ RNA required?
The RNA template may be a total RNA preparation, an mRNA [poly(A)+] population or a synthesized RNA transcript. Successful reverse transcription is dependent on the integrity and purity of the RNA used as a template. Regardless of the type of RNA used, it is critical that an RNase-free environment be maintained during RNA isolation. Additionally, care should be taken to minimize the amount of DNA present in the RNA preparation.
Q 05: How much RNA is required for the RobusT™ RT-PCR Kit?
The minimum amount of RNA that can be amplified using the RobusT RT-PCR Kit is dependent on many factors such as primer design, target size, and abundance of message. For the Positive Control RNA provided, the minimum amount of RNA required is 1 fg. Excellent amplification results can be obtained using total RNA template levels in the range of 1 pg -1 µg per reaction, or poly(A)+ RNA template levels in the range of 1 pg -100 ng. This system was able to detect ARF (human ADP ribosylation factor I), a low abundance message from as little as 100 pg of human skeletal muscle total RNA.
Q 06: What conditions may require optimization when using the RobusT™ RT-PCR kit?
There are several parameters which may be optimized for a particular primer-target combination including: MgCl2 concentration, primer annealing temperature and number of amplification cycles.
The optimal magnesium concentration is dependent on the final concentration of dNTPs, primers and template. Although 1.5 mM MgCl2 concentration is suitable for most applications, titration of the magnesium concentration for each experimental target/primer combination can significantly improve the sensitivity and specificity. To determine the optimal magnesium concentration for a specific application, prepare a reaction series containing 0.5-3.0 mM MgCl2 in 0.5 mM increments.
For primers with a high Tm, it may be advantageous to increase the suggested annealing and extension temperatures. The higher temperature minimizes nonspecific primer annealing thus increasing the amount of specific product produced.
Most RNA targets can be detected following 30 cycles of PCR amplification. However, if the target is rare or if only a small amount of starting material is available, it may be necessary to increase the number of cycles to 35 or 40.
Q 07: Why does the RobusT™ RT-PCR Kit use AMV Reverse Transcriptase rather than MMLV Reverse Transcriptase?
RNA transcripts exhibiting significant secondary structure must be denatured for efficient reverse transcription. In order to minimize secondary structure, the first strand cDNA synthesis must be carried out at an elevated temperature. While the maximum recommended temperature for MMLV Reverse Transcriptase is 42 °C, AMV Reverse Transcriptase is active at 60 °C in the optimized RobusT™ Reaction Buffer. Thus, the use of AMV Reverse Transcriptase eliminates many problems associated with secondary structure.
Q 08: What is the highest temperature that AMV Reverse Transcriptase can be used in?
For RT-PCR, AMV Reverse Transcriptase can be used up to 60 °C. For amplicons expected to be 1 kb or less even 65 °C can be used. Although AMV RT is rapidly inactivated at this temperature it remains active for the period of time that is necessary for transcription of short products. For PCR products expected to be greater than 1 kb a maximum first strand synthesis temperature of 60 °C is suggested. Be sure your first-strand primer anneals at the high temperature.
Q 09: How can I tell if my RT-PCR product is RNA specific?
Include a control reaction without reverse transcriptase (negative control). Note! DyNAzyme EXT DNA Polymerase exhibits weak reverse transcriptase activity so short target sequences can be reverse transcribed even in the absence of AMV.
Primers may be designed to anneal to sequences in two exons on opposite sides of an intron to differentiate between amplification of cDNA and contaminating genomic DNA. An amplification product from genomic DNA will be larger than the mRNA derived product that lacks the intron sequence. Alternatively, primers can be designed to anneal to the exon-exon boundary of the mRNA. With such primers, amplification of genomic DNA will be highly inefficient.
Q 10: What are the differences among RNase H, RNase A and RNase B?
The main difference between all RNases are where they cleave the RNA (what site they recognize) and whether it is single stranded or double stranded.
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.
RNase A is an endoribonuclease that specifically hydrolyzes RNA after C and U residues. Cleavage occurs between the 3'-phosphate group of a pyrimidine ribonucleotide and the 5'-hydroxyl of the adjacent nucleotide. The reaction generates a 2':3' cyclic phosphate which then is hydrolyzed to the corresponding 3'nucleoside phosphates.
RNase B is a glycoprotein which possesses an amino acid composition indistinguishable from that of RNase A and which contains carbohydrate to the extent of 6 residues of mannose and 2 residues of N-acetylglucosamine per molecule. It is consequently considered to be a carbohydrate derivative of RNase A (1). RNase B has the same specificity as RNase A (2).
(1) Tarentino, A., Plummer, J., and Maley, F.: Studies on the Oligosaccharide Sequence of Ribonuclease B, J. Biol. Chem., 245, 4150 (1970)
(2) Plummer, T., and Hirs, C.: The Isolation of Ribonuclease B, a Glycoprotein From Bovine Pancreatic Juice, J. Biol. Chem., 238, 1396 (1963)
