ProtoScript II First Strand cDNA Synthesis Kit features two optimized mixes,
ProtoScript II Enzyme Mix and ProtoScript II Reaction Mix. The enzyme mix
combines ProtoScript II Reverse Transcriptase and Murine RNase Inhibitor,
while the reaction mix contains dNTPs and an optimized buffer. ProtoScript
II Reverse Transcriptase is a recombinant M-MuLV reverse transcriptase
with reduced RNase H activity and increased thermostability. It can be used
to synthesize first strand cDNA at higher temperatures than the wild type
M-MuLV. The enzyme is active up to 48°C, providing higher specificity and
higher yield of cDNA.
The kit also provides 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
templates including both mRNAs and non-polyadenylated RNAs. The first
strand cDNA product generated is up to 10 kb.
Figure 1: cDNA Synthesis of Jurkat RNA with the ProtoScript II First Strand cDNA Synthesis Kit
First strand cDNA synthesis was carried out in the presence of 1X ProtoScript II Reaction Mix and 1X
ProtoScript II Enzyme Mix at 42°C using 250 ng of Jurkat total RNA. Four amplicons from different
genes were amplified using 1X LongAmp® Taq 2X Master Mix (NEB #M0287). Lane 1, a 1.9 kb
amplicon from SDHA gene; Lane 2, a 5.5 kb from the guanine nucleotide exchange factor; Lane 3,
a 7.3 kb amplicon from Xrn-1 gene; and Lane 4, a 9.2 kb amplicon from fibrillin gene. Marker M is
the 1 kb Plus DNA Ladder (NEB #N3200).Quantitative detection of reverse transcription products using the ProtoScript II First Strand cDNA Synthesis Kit
Ten-fold serial dilutions of in vitro transcribed luciferase RNA (1x102 to 1x109 copies) were mixed with 1 ng Jurkat total RNA. The RNA was reverse transcribed using first strand cDNA synthesis kits, as indicated, and 1/10th of the cDNA product was used for qPCR using luciferase-specific primers and iTaqTM Universal SYBR Green Supermix (BioRad). Ct values were plotted as a function of input luciferase RNA equivalent copies. Each data point represents the mean +/- 95% confidence interval of a minimum of 5 amplification reactions. ProtoScript II – NEB ProtoScript II First Strand cDNA Synthesis Kit and random primer mix; SuperScript II – Invitrogen SuperScript First Strand Synthesis System for RT-PCR and random hexamers; SuperScript® III – Invitrogen SuperScript III First Strand Synthesis SuperMix and random hexamers.
This product is related to the following categories:
Liao, J. and Gong, Z. (1997). Biotechniques. 23, 368-370.
Sambrook, J. and Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual, (3rd ed.),. (pp. 8.46–8.53 avnd 11.37–11.42). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Check the integrity of the RNA by denaturing agarose gel electrophoresis (2). RNA should have a minimum A260/A280 ratio of 1.7 or higher. Ethanol precipitation followed by a 70% ethanol wash can remove most contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides (2).
Phenol/chloroform extraction and ethanol extraction can remove contaminant proteins such as proteases (2).
Some target RNA may contain strong pauses for RT; Use random priming instead of d(T)23VN.
Use sufficient amount of RNA.
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The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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