Trizol rna extraction method

CHPV infected PBMC, CD3⁺, CD14⁺, CD19⁺, CD56⁺ and mock controls were harvested at different time intervals post infection. CD19⁺cells were treated with 0.01 % of sodium azide and infected with CHPV to analyze for replicative intermediate for ruling out the possibility of the uptake of CHPV was not due to capping of immunoglobulin. RNA was extracted from cell lysate by using TRIzol® RNA extraction method (Life Technologies) as per manufactures instruction. One step Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using kit containing 2X PCR mix and SuperScript® III Reverse transcriptase/Platinum^®Taq DNA polymerase (Invitrogen CA, USA). One microgram of total RNA was subjected to one step RT-PCR with different primer pairs corresponding to the following:
i) upstream (5’-CGG AGA GAA ATG TTG TTG TGT G-3’) and downstream (5’-GCA AAG AGT TTC CTG GCG TA-3’) primers specific for a 150 bps amplicon of the N gene, used for transcription assessment. For replication assessment ii) upstream (5’-TGG AAA GGG TAG GAG ATA TTC GA-3’) and downstream (5’-GAG AGT GTC CTG AAG CTT TGG-3’) primers specific for a 150 bps amplicon of the N-P gene junction specific primers were used [12 ]. The RT-PCR products were resolved on 1.8 % agarose gel and visualized by staining with ethidium bromide.

Total RNA from Eye-antenna imaginal discs containing wild-type or mutant clones was isolated using a Trizol RNA extraction method (Invitrogen). The SuperScriptIII First-Strand Synthesis System (Invitrogen) kit was used to synthesize complementary DNA. Real-time PCR was carried out on an Applied Biosystems machine using the SYBR green fast kit (Applied Biosystems) following the manufacturer's instructions. Relative gene expression was obtained from triplicate runs normalized to Rp49 as endogenous control. The following primers were used: Rp49, 5′-GGCCCAAGATCGTGAAGAAG-3′ and 5′-ATTTGTGCGACAGCTTAGCATATC-3′; Spitz, 5′-CGCCCAAGAATGAAAGAGAG-3′ and 5′-AGGTATGCTGCTGGTGGAAC-3′
Clones of mutant cells in the eye-antennal discs were generated using standard MARCM system56 (link). Immunostaining and Immunoprecipitation experiments were performed as previously described57 (link).