ProtoScript® First Strand cDNA Synthesis Kit features two optimized mixes, M-MuLV Enzyme Mix and M-MuLV Reaction Mix. M-MuLV
Enzyme Mix combines M-MuLV Reverse Transcriptase and Murine RNase Inhibitor,
while M-MuLV Reaction Mix contains dNTPs and an optimized buffer. The kit also
contains two optimized primers for reverse transcription and nuclease-free
water. An anchored oligo-dT primer [d(T)23VN] forces the primer to
anneal to the beginning of the polyA tail. The optimized Random Primer Mix
provides random and consistent priming sites covering the entire RNA template
including both mRNAs and non-polyadenylated RNAs. The first strand cDNA product
generated is more than 10 kb (Figure 1).
General Information for
Successful cDNA Synthesis:
Template RNA
Intact RNA of high purity is essential for sensitive RT-PCR detection. RNA
should have a minimum A260/A280 ratio of 1.7 or
higher.
Either total RNA or mRNA can be used in the reverse
transcription reaction. Total RNA is generally sufficient for most RT-PCR
analyses. However, if desired mRNA can be easily obtained using a PolyA Spin
mRNA Isolation Kit (NEB #S1560) or Magnetic mRNA
Isolation Kit (NEB #S1550).
The amount of
RNA required for detection depends on the abundance of the transcript of
interest. In general 1 ng to 1 μg total RNA or 0.1-100 ng mRNA are
recommended.
First Strand cDNA Synthesis
Reaction
Denaturation of RNA and primer at 70°C for 5 minutes can
remove secondary structures that may impede long cDNA synthesis. However, this
step can be omitted in some cases (unpublished results).
We recommend
incubation at 42°C for one hour for maximum yield and length. However, many
targets can be detected after a much shorter incubation time. For example, 5
minutes incubation is enough for a 2 kb cDNA synthesis.
Choice of
Primers for Reverse Transcription
Oligo d(T) priming is preferred
for most applications because it ensures that all cDNA copies terminate at the
3´ end of the mRNA and produces the longest contiguous cDNA. An anchored
oligo-d(T) primer [d(T)23VN] forces the primer to anneal to the start
of the polyA tail, thereby preventing priming at internal sites in the polyA
tail (1). However, two other priming choices are possible if desired.
The Random Primer Mix is an optimized mix of hexamer and
d(T)23VN primers. It provides random priming sites covering the
entire RNA templates including both mRNAs and non-polyadenylated RNAs (such as
ribosomal RNAs). The Random Primer Mix yields shorter cDNAs on average and can
be used for the detection of multiple short RT-PCR products. Random Primer Mix
offers good performance in a wide range of RNA templates.
When a
gene-specific primer is used in a cDNA synthesis reaction, the cDNA product can
be used only for amplification of that transcript. This priming method gives
good results when the amount of RNA is limiting (below 10 ng) and only one
particular cDNA is desired.
Recommended primer concentration:
PRIMER
-- Final conc.
OLIGO d(T)23VN -- -- 5 μM
RANDOM PRIMER MIX --
6 μM
SPECIFIC PRIMER -- 0.1–1 μM
Figure 1:
First strand cDNA synthesis was carried out with 1X M-MuLV Enzyme Mix at 42°C using 2 μg of human spleen total RNA. Negative control reactions were carried out with 1X M-MuLV Reaction Mix. A fraction of the first strand cDNA product was used to amplify sequences specific for three different messenger RNAs using 1X LongAmp™ Taq 2X Master Mix (NEB #M0287 ). Lane 1: 1.1 kb of beta-actin gene. Lane 2: noRT control of 1.1 kb of beta-actin gene. Lane 3: 4.7 kb of Xrn-1 gene. Lane 4: noRT control of 4.7 kb of Xrn-1 gene. Lane 5: 9.8 kb of guanine nucleotide exchange factor p532. Lane 6: noRT control of 9.8 kb of guanine nucleotide exchange factor p532. Marker M is 2-Log DNA Ladder (NEB #N3200 ).
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