Superscript reverse transcriptase kit

SuperScript™ reverse transcriptase kit (Invitrogen, USA) was used for RT-PCR reaction. Total RNA template was mixed in a 20μl reaction containing 50 ng of random hexamer primer, 10 mM dNTP mix, 4 μl of 5X First-Strand buffer, 1 μl of 0.1 M DDT, and 200 units of SuperScript™ RT. The IBDV hypervariable VP2 region was detected by using primer pair BG-VP2 designed using LaserGene software. The primer pair BG-VP2 produces a DNA amplicon of 699 base pairs. The visualization of the PCR product was done by electrophoresis on a 1.5% agarose gel stained with Ethidium Bromide [19 (link), 20 (link)].

Tissue or cells were lysed using 1 mL TRIzol reagent (Invitrogen). Then, 200 μL of chloroform was added and shaken to homogenize the lysates, which were then centrifuged at 12,000 × g for 10 min at 4 °C. The upper aqueous phase was collected, and an equal volume of 70% ethanol was added. Total RNA was extracted using the PureLink RNA Mini Kit (Invitrogen, 12183020). RNase-free DNase (QIAGEN, 79254) was added to reduce DNA contamination. cDNA was obtained by a reverse transcription reaction using the Superscript Reverse Transcriptase Kit (Invitrogen). The detection of cDNAs for the indicated genes was completed by qRT-PCR. qRT-PCR was conducted in the AGILENT MX3005P equipment using the SYBR method utilizing Power SYBR Green PCR Master Mix (ABI, 4367659). Samples were normalized to β-actin or U6 expression via 2^−ΔΔCT calculation. The detection of tRF-Met-CAT-049 and tRF-Gln-CTG-026 was performed by a specific reverse transcription primer. After RNAs were extracted, RNAs were reverse transcribed with a specific primer. The primers for the detected genes are listed in Supplementary Table S3. All kits were utilized in line with the manufacturers’ instructions.

Total RNA was extracted from heart tissue or CFs with TRIzol reagent (Invitrogen, CA, United States), and reverse transcribed with SuperScript reverse transcriptase kit (Invitrogen, CA, United States). Gene expression was analyzed by quantitative real-time polymerase chain reaction (PCR) using SYBR^® dye (LightCycler^® 96 Real-Time PCR System, Roche, Switzerland). Transcript quantity was determined by relative value to the reference 18S rRNA and normalized to the mean value of samples. The primers encoding Mus musculus angiotensinogen (AGT), ACE, AT1R, TGFβ1, and 18S were as follows:

The effect of phytochemicals on the expression of C. jejuni virulence genes was investigated using real-time quantitative PCR, as described previously (Upadhyay et al., 2012 (link)). Each C. jejuni strain was cultured separately with the higher SIC of phytochemicals at 37°C in CEB to mid-log phase (8 h) and total RNA was extracted using RNeasy RNA isolation kit (Qiagen, Valencia, CA). Complementary DNA was synthesized using the Superscript Reverse transcriptase kit (Invitrogen). The cDNA synthesized was used as the template for RT-qPCR. The primers for each gene (Table 1) were designed from published GenBank C. jejuni sequence using NCBI Primer design software. The amplification specificity was tested using NCBI-Primer BLAST, melt curve analysis and in silico PCR amplification (Bikandi et al., 2004 (link)). The amplified product was detected using SYBR Green reagent. Relative gene expression was determined using the comparative critical threshold (Ct) method on a Quant Studio 3 Real Time PCR system. Data were normalized to the endogenous control (16S rRNA gene), and the level of candidate gene expression between control and phytochemical treated sample was analyzed.

Skin lesion biopsy samples were collected from each CL patient and snap frozen in liquid nitrogen. Total RNA was extracted from these lesion samples and reverse-transcribed to cDNA using SuperScript Reverse Transcriptase kit (Invitrogen, Carlsbad, California). The primers and probes for detecting each cytokine cDNA were listed in Table 1. The housekeeper β-actin gene was added as control. Real-time quantitative PCR (RT-qPCR) was performed using Applied Biosystems 7500 Fast Real-Time PCR System. Comparative C_T (ΔΔCT) method was used to calculate differences in cytokine gene expression value compared to housekeeping β-actin gene as control.