Quantinova sybr green

RNA for qRT-PCR analysis was prepared from two independent experiments of infected RAW 264.7 cells at 3 and 6 h post-infection at MOI 10. Bacterial RNA was isolated using Ambion TRIzol reagent (Life technologies) and Direct-zol RNA Miniprep kit (Zymo Research). The cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer’s protocol (priming: 25°C, 5 min; reverse transcription: 42°C, 30 min; RT inactivation: 85°C, 5 min; and store temperature: 4°C). The concentration and purity of cDNA were measured and normalized to 100 ng/mL for qRT-PCR step. The primers for qRT-PCR indicated in Table 1 were designed and then evaluated for specificity by conventional PCR using Q5 High-fidelity DNA polymerase (New England Biolab). Gene expression was quantified using QuantiNova SYBR green (Qiagen) following the PCR cycling program as follow: initial heat activation step at 95°C for 2 min; two-step 40 cycles of 5 s at 95°C and 30 s at 60°C. The threshold cycle and melting curve of each gene were automatically established and recorded by the software CFX Maestro Software (version 4.0). Relative gene expression level of each gene in ΔbicA mutant was normalized to wild-type strain using the 2^−ΔΔCt method with 16S rRNA as reference gene.

Total RNA was extracted with TRIzol reagent (Catalog# 15596026, Invitrogen). One μg RNA was reversely transcribed using GoScript™ Reverse Transcription Mix from Promega (Catalog# A2801). Real-time Quantitative PCR (QPCR) was performed with ViiA 7 Real-Time PCR System (Applied Biosystems) using QuantiNova SYBR® Green (Catalog# 208056, QIAGEN) with the primers targeting different genes (Supplementary Table 2).

RNA was extracted from kidney tissue (n = 6) with the RNeasy Plus Mini Kit (Qiagen, CA, United States), and used to generate cDNA with the iScript cDNA Synthesis Kit (Biorad, CA, United States). The QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific) was used to perform RT-PCR. Mastermixes incorporated QuantiNova SYBR Green (Qiagen) and the primers listed in Table 1.

RNA was isolated from whole uteri tissue or cultured cells using the RNeasy Mini Kit (QIAGEN, 74104) according to manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA (100–250 ng) was reverse-transcribed and cDNA made using Superscript III first strand synthesis kit (Invitrogen, 18080051). Real-time qPCR was performed using QuantiNova SYBR Green (QIAGEN, 208052) with oligo primer pairs (MilliporeSigma; Supplemental Tables 4 and 5). Expression levels were normalized to housekeeping genes 18s (mouse) and β-actin or GAPDH (human) and analyzed using comparative cycle threshold (ΔΔCT) method as previously described (100 ). For each analysis, relative gene expression was calculated relative to the average ΔΔCT value for the control samples.

Total RNA was extracted from plant leaf samples using TRIzol™ Reagent (Invitrogen, USA) following the procedures provided by the manufacturer. HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, China) was applied to obtain cDNA samples to perform qPCR analysis with QuantiNova™ SYBR^® Green (Qiagen, Germany) and a Bio-Rad CFX96™ System coupled to a C1000 Thermal Cycler (Bio-Rad, USA). The reference gene Ubiquitin (UBQ) (LOC_Os03g13170) was used as an internal control. The primers used are listed in Additional file 3: Table S2.