Q1：Why is the CT value on the high side of the reverse transcribed cDNA product tested by qPCR?
A1：
1) Template RNA extraction failed or degraded. It is recommended to verify the integrity of RNA by electrophoresis before reverse transcription reaction, and perform reverse transcription and qPCR operations as soon as possible after RNA extraction.
2) The gene expression level is low. The amount of template RNA can be appropriately increased.
3) Improper operation of reverse transcription system, program or sample addition. It is recommended to operate according to the specifications of the manual.
Q2：Why is the melting curve not a single peak when performing qPCR after reverse transcription?
A2：
1) RNA template contains gDNA. Removal of the genome reaction (Cat. No.: SR511) or primer-spanning intron design before reverse transcription is recommended.
2) Poor specificity of qPCR primers. Redesign primers with better specificity, and the specificity of primers can be detected by conventional PCR.
3) Excessive primers. Excessive primers may form primer-dimers, and it is recommended to add them according to the amount recommended in the instructions.
4) The system may be polluted. Set up a no-template negative control (NTC) to check whether the system is contaminated. If there is contamination, it is recommended to replace the reagents and consumables, and use a nucleic acid cleaner (Cat. No.: ND709) to clean the work surface and aerosol contamination in the experimental environment.
