Primerscript rt reagent kit with gdna eraser

Four pear accessions (“Zaoshengxinshui,” “Qiushui,” “ZQ65,” and “ZQ82”) at six time points were used to extract RNA. At each time point, all accessions had three biological replications. A total of 72 samples were analyzed. First-strand cDNA was synthesized using the PrimerScript RT Reagent Kit with gDNA Eraser (RR047, Takara, Osaka, Japan). The real-time PCR mixture (10 μl total volume) contained 5 μl of TB Green Premix Ex Taq (RR420A, Takara, Osaka, Japan), 0.5 μl of each primer (10 μM), 0.5 μl of cDNA, and 3.5 μl of double-distilled water. The reactions were performed on a LightCycler 480 instrument (Roche, Basel, Switzerland), according to the manufacturer’s instructions. The two-step quantitative PCR (qPCR) program was initiated at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The expression was calculated as 2^−ΔΔCt and normalized to the expressions of the actin gene (JN684184) and UBI (XM_009368893.2). All the primers used are listed in Supplementary Table S1.

Total RNA was extracted by using Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. The quantification of the extracted RNA was measured by using NanoDrop 2000 spectrophotometer, and the integrity of the RNA was determined by 1% agarose gel electrophoresis (BIOWEST, Spain). Equivalent amount of the total RNA (1000 ng) from each sample was used for cDNA synthesis with PrimerScript™ RT reagent kit with gDNA Eraser (TaKaRa, Japan) for qPCR (Bio-Rad, USA) in a 20 μL reaction volume. The prepared cDNA templates were store at -20°C, and a 10-fold dilution was required in qRT-PCR. The qRT-PCR was performed on a 7500 Real-time PCR system (Life Technologies, USA). The PCR reaction volume was 20 μL, containing 10 μL 2 × TaKaRa Ex Taq™SYBR premix, 3 μL of diluted cDNA, 2 μL of each primer (2 μM), 2.6 μL DEPC treated water (Invitrogen, USA), 0.4 μL Rox Reference Dye II (Takara, Japan). The PCR program was 95°C for 3 min, 95°C for 15 s, 60°C for 1 min then go to step 2 and repeated for 40 cycles. CT values determined for each sample were normalized against the values for housekeeping gene β-actin. The results were further compared to respective control group to determine the change of gene expression by the 2^−ΔΔCt method (33 (link)). The primers used for qRT-PCR were listed in Supplemental Table 1.

Total RNA was extracted from the mouse tissues using TRIzol reagent (Invitrogen, Shanghai, China), and cDNA was synthesized using the PrimerScript RT reagent Kit with gDNA Eraser (Takara, Beijing, China, cat. #RR047). Real-time PCR was performed using the two-step quantitative RT-PCR method in accordance with the manufacturer’s instructions (Takara, cat. #RR820) on a QuantStudio 7 Flex Real-Time PCR System (ThermoFisher Applied Biosystems, Waltham, MA, USA). The expression level of each target gene was normalized to the sample’s Gapdh mRNA level and is expressed relative to the respective wild-type level. The following primer sets were used:
Slc39a14 forward 5′-
TTTCCCAGCCCAAGGAAG-3′ and reverse 5′-
CAAAGAGGTCTCCAGAGCTAAA-3′; Dmt1 forward 5′-
CGGGGATGAATGACTTCCTG-3′ and reverse 5′-
GGACATAAACCACTACAAAGTACA-3′; Slc39a8 forward 5′-
TGCCTGGATGATCACGCTTT-3′ and reverse 5′-
CGGGTGCTCATTCCTGCAT-3′; Slc30a10 forward 5′-
GCCACCTTGCACATCAAACA-3′ and reverse 5′-
GCTTCTTAGCGCAGCTCTGG-3′; Slc13a5 forward 5′-
CAGGGCTCTCGAAGTGGATG-3′ and reverse 5′-
GAATCATGACATACAGAGGATGGA-3′; Slc2a4 forward 5′-
AACGGGTTCCAGCCATGAG-3′ and reverse 5′-
AACCCATGCCGACAATGAAG-3′; Fpn1 forward 5′-
GTCGGCCAGATTATGACATTTG-3′ and reverse 5′-
ATTCCAACCGGAAATAAAACCA-3′.

Total RNA was isolated from tumor tissues Trizol Reagent (Invitrogen Life Technologies) according to manufacturer’s instructions. Primer Script RT reagent Kit with gDNA Eraser (Takara BIO INC, Kusatsu, Shiga,Japan) was used for reverse transcription of RNA. Real-time quantitative PCR was performed using the SYBR Premix Ex Taq RNAse H+ kit (Takara), and the results were analyzed using the Bio-RAD detection system (Bio-RAD, USA).

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. About 0.1 mg of total RNA was reversed to the first cDNA using the PrimerScript RT Reagent Kit with gDNA Eraser (Takara, Beautilion, Otsu, Japan). Primers used for qRT-PCR are listed in Supplementary Table 1. The quantified expression level of the target gene was normalized against the housekeeping gene, 18S rRNA (GenBank accession no. MF101220.1). ABI-7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) was applied for qRT-PCR using the SYBR Green master mix (Takara). The conditions for quantitative analysis were as follows: 94°C for 2 min; 35 cycles of 94°C for 10 s, 62°C for 15 s, and 72°C for 20 s; and a final extension at 72°C for 10 min. Gene expression was normalized with the 2^–ΔΔCt method setting HSM-cultured groups as controls.

Total RNA was extracted from skeletal muscle tissues using RNAiso (Takara, Tokyo, Japan) in accordance with the manufacturer’s instructions. The quality and purity of total RNA were assessed using a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA, USA) at 260 and 280 nm. The absorption ratios (260/280 nm) of all samples were between 1.80 and 2.00. cDNA was synthesized by the Primer Script RT Reagent Kit with gDNA Eraser (Takara, Tokyo, Japan). Messenger RNA levels were measured by qPCR using a Quant Studio 3 system (Thermo Fisher Scientific, Waltham, MA, USA). Gene-specific primers were designed online by Primer 3 (version 4.1.0) (https://primer3.ut.ee/ (accessed on 14 December 2021)) and are listed in Supplementary Table S2. The PCR protocol was as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Relative gene expression levels were calculated using the 2^−ΔΔCt (threshold cycle difference) method. GAPDH was used as the housekeeping gene.

Total RNA was extracted from N. benthamiana leaves using TRIzol reagent (Invitrogen, New York, NY, USA). After removal of genomic DNA with RNase-free DNase Ι (TaKaRa), first-strand cDNA was synthesized using the Primerscript RT reagent kit with gDNA Eraser (TaKaRa, Kusatsu, Japan). Quantitative real-time (qRT)-PCR was performed using a 2 × SYBR Premix Ex TaqTM (TaKaRa), and actin was used as the internal control. All qRT-PCR experiments were completed in triplicate using three independent samples [34 (link)].
N. benthamiana leaves were ground in protein extraction buffer (6 M urea, 1 mM EDTA, 50 mM Tris–HCl, 1% SDS, pH 7.5). Proteins were separated on 10% w/v SDS-PAA (Sodium dodecylsulfate (SDS)-polyacrylamide (PAA)) gel and transferred to polyvinylidene fluoride by semi-dry blotting and were detected with an antibody (Roche, Basel, Switzerland) [34 (link)].