We performed two nasopharyngeal (NP) and two mid nasal (MN) swabs on each of three subjects, and placed them in 500µL of TE or Cells-to-cDNA II lysis buffer. We used Copan swabs, cut the swab into an eppendorf tube, vortexed and placed at 4˚C. The SK NP and MN swabs in lysis buffer were heat inactivated at collection at 75˚C for 15 minutes. The rest we heat inactivated before measurement.
For measurement, we added water 1:1 v/v to the lysis buffer, and lysis buffer 1:1 v/v for the TE samples, and then heat inactivated at 75˚C for 15 minutes.
We did qPCR using two separate RNA molecules over the course of five days. The charts below give Cq values of the swabs over the time course (later time points look similar). The long and short of it is that all the samples remained relatively stable over time.
NOTE: The primers used for RPP3 amplification contain the Illumina adaptor sequences, explaining the high NTC values relative to the GAPDH sample. This is especially aggravated at high primer concentrations (400 nM used in this assay) and was a big motivator for us to minimize RPP3 primer concentrations in the seq assay.
15. In Lysate RT-qPCR of various swab collection techniques
17. [24 hr]In Lysate RT-qPCR of various swab collection techniques
22. [72 hr]In Lysate RT-qPCR of various swab collection techniques
23. [120 hr]In Lysate RT-qPCR of various swab collection techniques