How many PCR cycles do I need to use for blood RNA libraries prepared using Globin Block?
Typically, libraries prepared from blood total RNA using Globin Block require one additional PCR cycle compared to libraries made with the standard QuantSeq Kit reagents.
However, we strongly recommend performing a qPCR assay to determine the optimal number of PCR cycles for the library amplification. The PCR Add-on and Reamplification Kit V2 (Cat. No. 208) includes the PCR Mix “PM”, the PCR Enzyme Mix “PE” and “P7” and “P5” primers that are required for the qPCR assay. In addition, SYBR Green I Nucleic Acid Gel Stain (10,000X in DMSO, Sigma-Aldrich Cat. No. S9430) is also required (user provided).
Further details of the qPCR assay are provided in these FAQs: