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RT² Profiler™ PCR Array System Controls

The RT² Profiler™ PCR Arrays and the RT² RNA QC PCR Arrays include built-in positive control elements for the proper normalization of the data, for the detection of genomic DNA contamination, for the quality of your RNA samples, and for general PCR performance in your hands.

Housekeeping Genes:
Wells H1 through H5 contain the species-specific housekeeping gene panel listed in the product information included with each cataloged or custom PCR Array. Use the raw threshold cycle data of these genes to choose an appropriate factor to normalize your PCR Array data, either the Ct value of one housekeeping gene or the average Ct value of up to all five genes. The Ct value of the chosen genes should not differ by more than one (1) cycle across the compared samples.

Genomic DNA Contamination:
Well H6 contains a Genomic DNA Control (GDC) primer set that specifically detects non-transcribed genomic DNA contamination with a high level of sensitivity.
If the GDC threshold cycle value is greater than 35, then the level of genomic DNA contamination is too low to affect gene expression profiling results. No action is needed.
If the GDC threshold cycle value is less than 30, then genomic DNA contamination must be removed from the RNA sample, and the analysis must be repeated.
If the GDC threshold cycle value is between 30 and 35, then the expression profiling results may still be interpreted gene-by-gene with caution.
For each gene of interest (GOI), calculate the difference between the GDC and the GOI threshold cycle values.
If this value is six or greater, the data may be used without further validation.
If this value is less than six, the results should be validated with individual gene-specific RT² qPCR Primer Assays that include a no reverse transcription (NRT) control. Or, consider removing the genomic DNA contamination from the RNA sample and repeating the analysis.

Reverse Transcription Controls:
Wells H7 through H9 contain replicate Reverse Transcription Controls (RTC) to test the efficiency of the RT² First Strand Kit (330401) reaction with a primer set to detect template synthesized from the kit's built-in external RNA control. Any impurities that affect the reverse transcription of the external RNA control also affect the reverse transcription of experimental RNA.
A value of (CtRTC -CtPPC) less than five (5) indicates no apparent inhibition.
A value greater than five (5) provides evidence of impurities that inhibited the reverse transcription phase of the procedure. Double check the quality, purity, and integrity of the RNA samples.

Positive PCR Controls:
Wells H10 through H12 contain replicate Positive PCR Controls (PPC) to test the efficiency of the polymerase chain reaction itself using a pre-dispensed artificial DNA sequence and the primer set that detects it. Any impurities that affect the positive control PCR amplification also affect amplification of the gene-specific products of interest.
Values of CtPPC = 20 +2 on each PCR Array indicate a lack of PCR inhibitors. Higher values and more widely variable values across arrays indicate the presence of inhibitors with different concentrations in or effects on the samples. Double check the quality, purity, and integrity of the RNA samples.

The two sets of replicate control wells (RTC and PPC) on each RT² Profiler™ PCR Array also test for inter-well, intra-plate consistency.