Steponeplus real time pcr platform

RNA was harvested and isolated following the RNeasy MiniKit standard protocol (QIAGEN 74104). The concentration of RNA (ng/ $\mathsf{\mu }$ L) was determined by a NanoDrop ND-1000 analyser (Analytical Technologies, Pennsylvania, USA). qRT-PCR was performed by preparing a master mix containing 30% cDNA sample, 50% Fast SYBR Green Master Mix (Applied Biosystems 4385612), and 0.1 $\mathsf{\mu }$ M forward and reverse oligo-primers, which were plated in triplicate. qRT-PCR reactions were run using the StepOnePlus Real-Time PCR Platform (Applied Biosystems, Foster City, CA, USA), initially at 95 $℃$ for 20 s, then 40 cycles of 95 $℃$ for 3 s and 60 $℃$ for 30 s. Data were processed using the comparative CT method, relative to the housekeeping gene NONO (Table 2). Changes in mRNA expression were expressed as Log2 fold change relative to DMSO controls and analysed using a Students t-test or one-way ANOVA (p < 0.05).

Samples with positive ELISA results that were subsequently confirmed by INNO-LIA were subjected to qPCR using the TaqMan system (Applied Biosystems, Foster City, CA) on the Applied Biosystems StepOne Plus Real Time PCR platform. The human albumin gene was used as an endogenous reaction control, and the viral gene regions pol (186 bp) of HTLV-1 and tax (75 bp) of HTLV-2 were used as viral confirmation and molecular differentiation targets (26 (link)). Each reaction contained 12.5 μL of TaqMan Universal PCR Master Mix (2X) (Applied Biosystems, Foster City, USA), 6.0 μL of ultrapure water, 0.5 μL of each primer, 0.5 μL of each probe and 5.0 μL of DNA, resulting in a total volume of 25 μL. The following temperature cycles were used: 95 °C for 10 mins, followed by 45 cycles of 95 °C for 15 secs and 60°C for 1 min for primer and probe binding.
The following primers were used in the reactions: 5′-CCCTACAATCCAACCAGCTCAG-3′ (HTLV-1F), 5′-GTGGTGAAGCTGCCATCGGGTTTT-3′ (HTLV-1R), 5′-CGATTGTGTACAGGCCGATTG-3′ (HTLV-2F), 5′-CAGGAGGGCATGTCGATGTAG-3′ (HTLV-2R), 5′-GCTGTCATCTCTTGTGGGCTGT-3′ (Albumin F), and 5′-AAACTCATGGGAGCTGCTGGTT-3′ (Albumin R). The probe sequences were as follows: FAM-5′- CTTTACTGACAAACCCGACCTACCCATGGA-3′-MGB (HTLV-1), FAM-5′- TGTCCCGTCTCAGGTGGTCTATGTTCCA-3′-MGB (HLTV-2) and FAM-5′- CCTGTCATGCCCACACAAATCTC-3′-MGB (Albumin) (26 (link)).

For the real time quantitative RT-PCR, 1 μg of total RNA was used to synthesize the cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Quantitative PCR was performed using the FastStart Universal SYBR Green Master (Roche) according to the manufacturer's instruction on the StepOne Plus Real time PCR Platform (Applied Biosystems). The qRT-PCRs were carried out with the following protocol: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and at 60°C for 60 s. The BrActin was used as the internal control, which has been shown to be one of the reference genes in Chinese cabbage. After the amplification, the melting curve was determined for specific product. Three independent biological replicates for each sample and three technical replicates for each biological replicate were analyzed. Significant differences of gene expression level between “GHA” and “XK” were evaluated using a student's t -test. All of the primers for the selected genes are listed in Table S1.

The qPCR reactions were carried out using the SensiFAST SYBR Hi‐ROX Kit (Bioline) on the StepOnePlus Real‐Time PCR platform (Applied Biosystems). To measure the transcriptional level of CRISPR‐Cas genes, mRNA was extracted from bacteria grown to exponential phase (A600 nm ~2.0) or post‐exponential phase (A600 nm ~4.0) using the PureLink RNA Mini Kit (Life Technologies). The mRNA was then reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis Kit (Life Technologies). In this set of qPCR experiment, genomic DNA was used in the standard curve and the 16S rRNA gene was used as an internal reference. To measure the frequency of the episomal LME‐1, a set of primer pairs were used to amplify from the genomic DNA extracted from bacteria before or after infection. In this set of qPCR experiment, plasmids containing each amplification product were used in the standard curve. The LME‐1 excision frequency and episomal copy number are calculated as
$\begin{array}{l}\text{Freq}\left(\text{Excision}\right)=\frac{\text{Amplicon}\left(\text{chrF}\&\mathrm{\text{chrR}}\right)}{\text{Amplicon}\left(\text{chrF}\&\text{chrR}\right)+\left[\text{Amplicon}\left(\text{chrF}\&\text{lmeR}\right)+\text{Amplicon}\left(\text{lmeF}\&\text{chrR}\right)\right]/2}\\ \text{CopyNumber}\left(\text{Episome}\right)=\frac{\text{Amplicon}\left(\text{lmeR}\&\text{lmeF}\right)}{\text{Amplicon}\left(\text{chrF}\&\text{chrR}\right)}\end{array}$