Transscript all in one first strand cdna synthesis supermix for qpcr one step gdna removal

Various floral organs including lemmas, paleae, stamens and pistils of NIP and Oat-like rice, and grains of NIP, Oat-like rice, OsMADS1^Olr-overexpression and OsMADS1-RNAi plants were collected from field-grown rice plants and subsequently stored at − 80 °C after freezing in liquid nitrogen. Total RNA was extracted from all samples using the RNA prep pure Plant kit (TransGen Biotech, Beijing, China). First-strand cDNAs were synthesized from 2 μg total RNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (TransGen Biotech, Beijing, China). The qRT-PCR was performed using SYBR Green Mix (CWBIO, Beijing, China) and run on a CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc. USA). The OsActin gene in rice was used as an endogenous control. The primers used in the qRT-PCR analysis are listed in Additional file 1: Table S6. To differentiate between the mutated OsMADS1^Olr and wild-type OsMADS1 transcripts by a combined RT-PCR amplification and sequencing method, cDNA fragments spanning the mutation site in OsMADS1^Olr or the corresponding wild-type site in OsMADS1 were amplified respectively from NIP, OE-2 and OE-10 plants with a designed OsMADS1-RT primer pair (Additional file 1: Table S5), and sequenced subsequently.

After the stress treatment, the mycelia were quickly scraped, blot-dried and stored in a −80 °C freezer. The RNA was extracted using an E.Z.N.A.™ Plant RNA kit (Omega Bio-Tek, Norcross, GA, USA). The first strands of cDNA were synthesized using TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (TransGen Biotech, Beijing, China). Real-time fluorescent quantitative PCR (RT-qPCR) was carried out using TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China) on a CFX96 real-time fluorescence quantitative PCR machine (Bio-Rad, Hercules, CA, USA). The level of expression of the untreated control was used as the reference for calculating the relative expression levels using the 2^−ΔΔCt method [58 (link)]. Glyceraldehyde-3-dehydrogenase (GAPDH) and 18S ribosomal RNA gene (18S rRNA) were used as reference genes. The PCR primers are shown in Table 3.

Total RNA from the seeds was isolated using the RNAprep Pure Plant Kit (Polysaccharides and Polyphenolics, Tiangen, Beijng, China) according to the manufacturer’s instructions. Three duplicates of total RNA extracts were reverse transcribed for first-strand cDNA synthesis for qRT-PCR using the TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (TransGen Biotech, Beijing, China). The qRT-PCR, performed by TransStart^® Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) with the total system was 20 μL, including 2 μL cDNA. The qRT-PCR conditions followed the manufacturer’s instructions. The primers were designed using Primer 6.0 software. The thermal cycling protocol was 30 s at 94 °C, then 40 cycles of 94 °C for 5 s and 60 °C for 30 s for annealing and extension. The specificity was assessed by the melt curve and size estimation of the amplified product. The expression levels of the DEGs were determined using the 2−∆∆t method. The reference large subunit ribosomal protein gene used for normalization of the assayed genes was Che034253 because its expression is stable throughout seed germination. The results shown are the average of three independent biological replicates, repeated three times.

The mRNA levels of specific gene mRNAs were assessed using RT-qPCR, as described by Cai et al. [52 (link)]. Briefly, total RNA from 100 mg of T. reesei mycelia was extracted using a FastRNA Pro Red Kit (MPbio, Irvine, CA, USA) according to the manufacturer’s instructions. For reverse transcription, we used TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (TransGen Biotech, Beijing, China) was used to reverse-transcribe 800 ng of total RNA to produce cDNA. RT-qPCR was performed using PerfectStart™ Green qPCR SuperMix (TransGen Biotech) with 1 μL of cDNA and 200 nM of the forward and reverse primers in a final volume of 20 μL. The sequences of the primers used in the RT-qPCR analysis are shown in Additional file 10: Table S4. For gene transcription analysis, the sar1 gene was used as the reliable reference in SYBR Green assays as previously described [57 (link)]. For RT-qPCR analysis, thermocycling was performed using an ABI StepOne thermocycler (Applied Biosystems, Foster City, CA, USA).

Human foreskin fibroblast cells were cultured in 12-well plates and infected with the RH or CEP tachyzoites at Multiplication of Infection (MOI) three for 1 h or 24 h, or left uninfected. The TRIzol^TM Reagent (Invitrogen, Thermo Fisher Scientific, Shanghai, China) was used for total RNA extraction from HFFs (1.0 × 10⁶ cells per well), and cDNA was synthesized with Random Primer (N9) using TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (Transgen Biotech, Beijing, China) and subjected to Real-time PCR with the primers shown in Supplementary Table 1. For protein level detection, HFF cells were harvested and subjected to Western blotting.