Lightcycler480 2 sequence detection system

To analyze the relative expression patterns of PvWOX3a in switchgrass, total RNA was extracted from E4I2 (Internode 2 at the E4 stage), E4L2 (Leaf 2 at the E4 stage), E3I2, E3L2, inflorescence, and crown bud tissues using a TRIzol Kit (TransGen Biotech, Beijing, China), and reverse transcribed into cDNA with a PrimeScript^TM RT Kit (TransGen Biotech, Beijing, China) after treatment with gDNA Eraser (Takara, Dalian, China). SYBR Premix ExTaq^TM (Takara, Dalian, China) was used for qRT–PCR, and the cycle thresholds were determined using a Roche LightCycler^® 480 II sequence detection system (Roche, Shanghai, China). The data were normalized to the level of PvUbq2 transcripts (GenBank accession NO: HM209468). In addition, the top two internodes of control and transgenic plants were used for total RNA extraction when control plants reached the E5 stage. qRT–PCR was used to validate the RNA sequencing results and for further analysis of differentially expressed genes among control plants and transgenic plants. The primers used for qRT–PCR are listed in Table S3.

Total RNA was isolated from cells in 96-well plates using the Qiagen FastLane Cell Probe Kit (QIAGEN, 216413), according to the manufacturer's instructions to a final volume of 40 μL per well, at the indicated timepoint after treatment. Gene expression was evaluated by qPCR using the ONE-step QuantiTect Probe RT-PCR Kit (Qiagen, catalog no./ID: 204445). For each reaction, 2 μL of RNA were used. Real-time qPCR reactions were performed on a Roche Lightcycler 480 II Sequence Detection System.
The following Taqman probes were obtained from Thermo Fisher Scientific: BRCA2 (Hs00609073_m1), IPO8 (Hs00914057_m1), RAD51B (Hs01568768_m1), RAD54L (Hs00941668.m1), Actin (Hs01060665.g1).
For data analysis, ΔC_t was calculated by subtracting average C_t of housekeeping genes from each C_t. An average ΔC_t for the control group was calculated and subtracted from ΔC_t to calculate negative ΔΔC_t. 2^−ΔΔC_t was used to calculate fold change.

Quantitative real-time RT-PCR was carried out as previously described with minor modifications (14 (link)). Briefly, total RNA of 1 µg was reversely transcribed in a 20 μl of reaction using Evo M-MLV RT Premix for qPCR (AG, Changsha, China, code no: AG11706) according to the manufacturer’s protocol. The reaction products were then diluted with 40 μl of distilled water. The real-time PCR reaction was used of 2 μl of diluted reverse transcription product, 10 µl of 2× SYBR^® Green Pro Taq HS Premix (SYBR^® Green Premix Pro Taq HS qPCR Kit, code no: AG11606), and 0.6 µl of forward and reverse primers (0.3 μM). The reaction was performed in a Light Cycler@ 480 II Sequence Detection System (Roche, Basel, Switzerland) for 45 cycles (95°C for 30 s and 60°C for 5 s) after an initial 30-s denaturation at 95°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The RNA levels of tumor samples and paired adjacent samples were calculated using the 2^−ΔCt method. All primers sequences of the hub genes and GAPDH were listed in Supplementary Table 1.

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was performed using One-Step RT-PCR System (Invitrogen). SYBR Green Real-time PCR Master Mix (Invitrogen) and Light Cycler 480 II Sequence Detection System (Roche) were used for qRT-PCR, and mRNA levels were normalized to β-actin. The sequences of the primers used for qRT-PCR are listed in Supplementary Table 1.

The mRNA was quantified by real-time PCR using a LightCycler480 II Sequence Detection System (Roche, Basel, Switzerland). All data were analyzed using β-Actin gene expression and GAPDH as internal standard. The sequences of primers used are available in supplemental table 1.

The total RNA of Min6 and INS-1 cells were extracted in TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s protocol. After spectrophotometry quantification, 1 μg total RNA was used for reverse transcription in a 20 μl final volume with AMV Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Quantification of mRNA by real-time PCR was performed using a LightCycler480 II Sequence Detection System (Roche, Basel, Switzerland). The reaction system (20 μl) contained the corresponding cDNA, forward and reverse primers, and SYBR GREEN PCR Master Mix (Applied Biosystems). The specific primers were as follows: 1) MafA (rat), 5′-AAGGAGGAGGTCATCCGACT-3′ (forward) and 5′-TCTGGAGCTGGCAC TTCTCG-3′ (reverse); 2) MafA (mouse), 5′-AAGCGGCGCACGCTCAAGAA-3′ (forward) and 5′- GGTCCCGCTCCTTGGCCAGA-3′ (reverse); 3) MR, 5′- AGACAATAGTCGGTCTGGGATT-3′ (forward) and 5′- GCTCAGGCTTCCTTGTTGGT-3′ (reverse); and 4) GR, 5′-AATGGGCAAAGGCGATAC-3′ (forward) and 5′-CAGGAGCAAA GCAGAGCAG-3′ (reverse). All data were analyzed using β-actin gene expression as an internal standard.

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen) and quantified with Nanodrop 2000 (Thermo Fisher Scientific). Complementary DNA synthesis was performed using total RNA and first stand cDNA synthesis kit using random primers (Roche). Real-time PCR was performed using the SYBR Green PCR Master Mix (Vazyme Biotech Co., Ltd., China) and LightCycler480 II Sequence Detection System (Roche). Relative mRNA levels were calculated using the 2^–ΔΔCt method and normalized to the expression of β-actin. The primers sequences were shown as follows: 5-HT_2CR froward (5′-GTT​CAA​TTC​GCG​GAC​TAA​GG-3′) and reverse (5′-TCA CGA ACA CTT TGCTTT CG-3′), Ins-1 forward (5′-CAC​TTC​CTA​CCC​CTG​CTG​G-3′) and reverse (5′-ACC​ACA​AAG​ATG​CTG​TTT​GAC​A-3′), Ins-2 forward (5′-GC TTCTTCTACAC ACCCATGTC-3′) and reverse (5′-AGC​ACT​GAT​CTA​CAA​TGC CAC-3′), β-actin forward (5′-AGG​CCA​ACC​GTG​AAA​AGA​TG-3′) and reverse (5′-AGAGCATAGCCC TCGTAGATGG-3′).