Rneasy minikit procedure

RNA isolation from the splenocytes was performed using the RNeasy minikit procedure (Qiagen).¹⁵ (link) Forward and reverse primers were used to amplify the transcripts. Samples (1 μg) of RNA from different experimental groups of mice were first utilized for cDNA synthesis with reverse primers (IDT; Sigma) using Mouse Moloney Leukemia Virus reverse transcriptase (Invitrogen) at 37 °C for 90 min. A common master mixture containing deoxynucleoside triphosphates (Promega) and Taq polymerase (Invitrogen), as well as gene specific primers and 0.25 volume of cDNA, was used for amplification with an Applied Biosystems thermocycler. The cycling conditions for genes of interest were 5 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 40 s, and extension at 72 °C for 40 s. The identities of the PCR amplified gene products were verified by agarose gel electrophoresis. Identical aliquots were processed in parallel without reverse transcriptase to rule out the presence of residual genomic DNA contamination in PCR amplification preparations. Densitometric analyses were done using the Quantity One software (Bio-Rad), ethidium bromide staining, and visualization under a UV transilluminator. For densitometric calculations, the same band area was used to determine band intensity and normalized for HGPRT.