Table 1.
Quantitative RT-PCR specifications
| Sample/Template | details |
|---|---|
|
| |
| Source | mouse cerebellum |
| Method of Preservation | dry ice/RNAlater-ICE |
| Storage time | <6 mo prior to isolation |
| Handling | Fresh |
| Extraction Method | TriZol |
| RNA: DNA-free | Intron-spanning primers, DNAse step |
| Concentration | Nanodrop |
|
| |
| Assay optimisation/validation | |
|
| |
| Accession number | Calb: Mm00486645_ml |
| Actin: Mm00607939_sl | |
| Amplicon Results | Calbindin: Chr.4: 15808411 – 15833856 (length 124) |
| Actin: Chr.5: 143664795 – 143668403 (length 115) | |
| Priming conditions | Oligo-dT |
| PCR efficiency | Calb: 93.6; Actin: 87.6 |
|
| |
| RT/PCR | |
|
| |
| Protocols | in text |
| Reagents | in text |
| NTC | undetectable |
| NAC | undetectable |
| Positive controls | none |
|
| |
| Data Analysis | |
|
| |
| Specialist software | calibrator method; BioRad CFX Manager Version 1.6 |
| Statistical justification | biological replicates |
| Transparent, validated normalisation | normalization to actin |