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By Nathan Rotenstreich
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In each case the total RNA sample may be spiked with 20fg cRNA. The usual control reaction ofPCR without RT can be carried out here to verify the absence of contaminating genomic DNA in the RNA preparations. Contamination may also be evident during amplicon separations, since most of the primers surround splice sites in the cellular targets, but some of the exons are very large. ll) of total RNA (SOng), and dT I 2-18 (lOOng) was heated for 15 min at 65°C. ll, BRL) were added and the solution heated for Ih at 37°C.
Pascale Legoux and Adrian J. Minty. Standardisation oln/RNA Quantification 32 range from 400 to 1600nt, with the great majority being less than 900nt. Outstanding exceptions are c-jun (2100nt), Krox20 (2600nt) and CSF-l (3600nt). The maximum distance in the controls is 600 nt. Ali to the second point, our experience with preparing cDNA libraries has shown us that Superscript reverse transcriptase is highly efficient at producing full-length transcripts from mRNA up to at least 7kb in length. If random priming with hexamers is chosen for the reverse transcnptlOn, it must be remembered that the whole RNA population will be transcribed, not just the 1-2% polyN RNA, possibly resulting in increased non-specific amplification.
Minty, Standardisation ofmRNA Quantification allowing one to use identical PCR conditions for each target. Wherever possible, the primers surround splice sites to avoid amplifYing contaminating genomic DNA and to help its detection. g. 410 bp in pQB-3. Exactly the same priming sites are found in the respective mRNAs, but the distance between them differs from that in the standard. This size difference facilitates separation of the amplicons after PCR. The constructs are contained in a pTZ plasmid, between a T7 promoter and a polydA stretch ending in a unique EcoRI site.