Hifi mmlv cdna kit

Total RNA in lung samples was extracted with the ultrapure RNA extraction kit (Cat# CW0581, CWbio Co., Ltd.) and was reverse-transcribed into cDNA using a HiFi-MMLV cDNA kit (Cat#CW0744, CWbio Co., Ltd.). The RNA expression levels were detected by Ultra SYBR Mixture (with ROX) (Cat#CW0956, CWbio Co., Ltd.) according to the manufacturer's instruction. PCRs were performed in Line Gene type 9600 Plus Real-Time PCR system (Bioer Technology Co. Ltd., Hangzhou, China). The primer sequences were as follows: β-actin (FW 5′-GCCTTCCTTCTTGGGTAT-3′ and RV 5′-GGCATAGAGGTCTTTACGG-3′) and KGF (FW 5′-TGCTTCCACCTCGTCTGTC-3′ and RV 5′-TCCTTCCATGTAGTCATAACTTCTG-3′). The amplification program was 95°C for 10 min followed by 45 cycles of 95°C for 15 s and 60°C for 60 s. The quantity of specific mRNA was normalized to the expression level of internal control β-actin mRNA. Gene expression was calculated using the 2^ΔΔCt method.

RT-PCR was undertaken to test expression of the vgb gene between NK-1 and NK-PV strains. Cells were harvested for RNA extraction after 16 h of cultivation in LB medium. The commercial RNApure Bacteria kit (Cwbio, Beijing, China) was used to extract total RNA. cDNA was obtained by reverse transcriptional synthesis with a HiFi-MMLV cDNA kit (Cwbio). Primers VG-F and VG-R were used to determine gene transcription.

Total RNA was extracted from fresh-frozen neural tissue using an Ultrapure RNA Kit (CWbio Co. Ltd., China) and then reverse-transcribed with a HiFi-MMLV cDNA Kit (CWbio Co. Ltd., China). Real time PCR was performed on a Bioer line gene PCR instrument (BIOER, China) using Invitrogen primers. The PCR amplification procedure includes 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 59°C for 60 s. Data were normalized to Beta actin.

The RNA extraction and real-time PCR was conducted according to the method described by Nasiri-Ansari et al. [26 (link)]. The total RNA in liver and small intestine was extracted with TRIzon reagent (CW0580, Cwbio, Beijing, China), and its concentration was determined by ultra-low volume spectrometer (BioDrop μLite. Biochrom Ltd. Shanghai, China). The RNA samples were immediately reverse transcribed into cDNA by a HiFi-MMLV cDNA Kit (CW0744, Cwbio, Beijing, China). The quantitative real-time PCR (RT qPCR) was conducted in a Real-Time PCR Detection System (CFX96, Bio-Rad, Hercules, CA, USA). BeyoFast™ SYBR Green qPCR Mix (2X) (D7260-5 mL, Beyotime, Shanghai, China) was used to amplify the target fragment. The primers used in this study and their target genes are listed in Table 1. The PCR procedure used in this study was as follows: pre denaturation (95 °C) for 2 min, followed by 40 cycles, denaturation (95 °C) 15 s, annealed (55 °C) 15 s and extension (72 °C) 30 s. At the end of the PCR procedure, the melting curve was observed at 65 °C to 95 °C to evaluate the specificity of the product. β-actin gene was used as housekeeping gene. The relative expression level of genes were normalized to that of β-actin and calculated according to the 2^−ΔΔCT approach.