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Griffin:Nested RT-PCR: Difference between revisions
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→-/+ Reverse Transcriptase
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====-/+ Reverse Transcriptase==== | ====-/+ Reverse Transcriptase==== | ||
RNA quality (purity and integrity) and quantity are critical to reliable gene expression analysis. | |||
Negative Reverse Transcriptase (-RT) as a control when preparing cDNA from an RNA sample extraction preparation. | |||
Reaction 1 (-RT) = Negative RT Control = RNA (DNase treated) + dNTPS + Random Hexamers/Oligo dT + RT Buffer + Water // NO Reverse Transcriptase//. | |||
Reaction 2 (+RT )= WITH Reverse Transcriptase (cDNA from polydt) | |||
Perform PCR with housekeeping primers. | |||
No gDNA contamination / -RT will be negative (no amplicon) / +RT will be positive | |||
YES gDNA contamination/ -RT will be positive (yes amplicon); incomplete DNase digestion of the RNA prep. | |||
Performing amplification of a sample prior to reverse transcriptase / minus reverse transcriptase control (no reverse transcriptase) determines to what extent genomic DNA contamination exists from the RNA prep. Determination of DNA contamination present in an RNA preparation controls for false positive qPCR. Amplification of traditional housekeeping genes (ie GAPDH) within a RNA prep (no RT) suggests gDNA contamination. | Performing amplification of a sample prior to reverse transcriptase / minus reverse transcriptase control (no reverse transcriptase) determines to what extent genomic DNA contamination exists from the RNA prep. Determination of DNA contamination present in an RNA preparation controls for false positive qPCR. Amplification of traditional housekeeping genes (ie GAPDH) within a RNA prep (no RT) suggests gDNA contamination. | ||
