Superscript 3 one step rt pcr system with

For further analysis, all positive samples were subjected to one-step RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit, Invitrogen, Carlsbad, CA, USA. In this procedure, we combined both cDNA synthesis (complementary DNA) and PCR amplification in a single tube and used genome-specific primers for all virus fragments: MBTuni-12 [5-ACGCGTGATCAGCAAAAGCAGG] and MBTuni-13 [5-ACGCGTGATCAGTAGTAGAAACAAGG]. Subsequently, the isolated fragments were cloned into the TOPO vector using the TOPO XL-2 Complete PCR Cloning Kit from Invitrogen and then sequenced with M13 primers using the Sanger method. The Sanger reads were assembled with the SnapGene v.2.3.2 software. For segments longer than the Sanger reads, sequencing was performed in several steps. After sequencing with M13 primers, the primers were synthesized for the further sequencing of the remaining part of the segment.

For characterizing the effect of freeze thaw on cell free mRNA expressions, RNA was extracted using plasma processed with S1, S2, S1FR, S2FR, and S1FRS2 conditions. Cell free mRNA was isolated by using plasma/serum circulating and exosomal RNA purification Kit (Norgen Biotek, cat. 42800) followed by 10X Baseline-ZERO DNase treatment (Epicentre, cat. DB0715K). DNase treated RNA samples were purified and further concentrated using RNA clean and concentrator (Zymo Research, cat. R1014). The purified RNA samples were assayed by RT-qPCR using custom selected 16 primers targeting MTND2, PPBP, B2M, PF4, ACTB, CORO1C, GSE1, GAPDH, SMC4, HBG1, NUSAP1, MIKI67, FGB, APOE, FGG, and ALB. Template RNA was mixed with Superscript III One-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen, cat. 11-732-020) to generate cDNA according to the protocol. PCR amplification products were treated with Exonuclease I (New England Biolabs, cat. M0293L) to digest single stranded primers at 37 °C for 30 min followed by inactivation of enzymes at 80 °C for 15 min. For RT-qPCR, cDNA from preamplification was diluted 1:80 and set-up in 96-well plates with SsoFast EvaGreen supermix with low ROX (BioRad, cat. 1725211) with above primers at 10 µM. QuantStudio 7 Flex (Applied Biosystems) was used to run RT-qPCR assay according to manufacturer’s recommended cycling conditions.

To check if the NDV detected in the oropharyngeal and cloacal swabs was the vaccine virus or the challenge virus, a fragment of the F gene was amplified and sequenced. We selected five oropharyngeal and six cloacal swab samples from the LaSota (live) booster-vaccinated chickens (Group A1) for sequencing. A 953 bp fragment from the 5′ end of the F gene was amplified using primer pairs NDVF13-F1 5′-GAC GCA ACA TGG GCT CCA RAY CTT-3′, NDVF13-R1 5′-GGC AAA CCC TCT GGT CGT GCT YAC-3′ as described previously [8 (link)]. The SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase (Invitrogen, Waltham, MA, USA) was used following the manufacturer’s instructions. The RT-PCR amplicons were purified using the FavorPrep™ GEL/PCR Purification Kit (Favorgen Biotech Corp., Ping Tung, Taiwan) and sequenced commercially (1st BASE, Selangor, Malaysia). The sequences were edited and analyzed using Bioedit and MEGA7 (www.megasoftware.net) software. Further, the complete F gene sequence of the LaSota vaccine strain was also obtained, as described previously [8 (link)]. The complete F gene sequence of BD-C161/2010 was retrieved from GenBank (Accession number MK934289.3).

A 25 µL reaction was prepared containing 5 µL of RNA, as described previously [31 (link)]. Briefly, 12.5 µL of 2× reaction buffer from the Superscript III one-step RT-PCR system with Platinum Taq Polymerase (Invitrogen, Carlsbad, CA, USA), 1 µL of reverse transcriptase/Taq mixture from the kit, 0.4 µL of a 50 mM MgCl₂ solution (Invitrogen, Carlsbad, CA, USA), 1 μL each of 10 μM forward and reverse of EMC-Orf1a primers with 400 nM concentration in the final solution, as well as 0.5 μL of 10 μM EMC-Orf1a probe with 200 nM concentration in the final solution was used. Molecular grade deionized water was used to make the final volume to 25 μL. Thermal cycling involved reverse transcription at 55 °C for 20 min, followed by denaturation at 95 °C for 3 min, and then 45 cycles of denaturation and extension at 95 °C for 15 s, and 58 °C for 30 s.

Hippocampal tissue samples were collected and RNA was isolated using the SV total RNA isolation system (Promega). The RNA was then used for the reverse transcriptase PCR reaction using the Superscript III one-step RT-PCR system with platinum Taq DNA Polymerase (Invitrogen) and the primers listed in Table 1 (Integrated DNA Technologies). The gene expression analyses were done using the comparative ΔΔCt method. The mRNA level changes were expressed as fold change as compared to the sham animals. All Ct values for target genes were normalized to CypA gene [49 (link)].