Primerscripttm rt reagent kit

To distinguish the expression of miRNA targets and the possibility of discovering new miRNA target genes under salinity, Cd, heat and drought stress in safflower, the expression levels of the miRNA-associated predicted sequences were assayed with qRT-PCR. Primers were designed for target and reference genes using Primer3, in accordance with the criteria described by Thornton and Basu [84 ] (S4 Table).
Total cDNAs synthesis was carried out with 1μg RNA using PrimerScript^TMRT reagent kit (TAKARA, Japan) according to the manufacturer’s instructions. The qRT-PCR was performed using gene-specific primers in a total volume of 10 μL as follows: 5 μLSYBR PremixExTaq^™ (Takara, Japan), 0.2 μL Reference Dye II (50X), 0.2 μL of each specific primers, 1 μL of the cDNAs as a template. Reaction conditions were as follows: 95°C for 5 min, 40 cycles at 95°C for 10 s, 55°C for 30 s, and 72°C for 30 s, followed by a final 10 min extension at 72°C and then was done disassociation stage (melting curve analysis).

Total RNA was isolated from gill tissue using sqRT-PCR RNAiso Plus (TaKaRa, Dalian, China). The cDNA was synthesized using the Perfect Real Time version of the PrimerScriptTM RT reagent kit with gDNA Eraser (Perfect Real Time) (TaKaRa,) according to manufacturer’s instructions. Then, sq-RT-PCR were chosen to analyze genes, and performed in a total reaction volume of 25 μl according to the manufacturers’ instructions. The S. paramamosain beta-actin gene and 18S ribosomal RNA gene were selected as the internal control. Primers used in this study are listed in Additional file 1: Table S1.

Total RNA of the collected aphid samples was isolated using TRIzol reagent (Life Technologies) and the first-single-strand cDNA was prepared using the PrimerScript^TM RT Reagent Kit (TaKaRa Bio, Dalian, China) according to the manufacturer’s protocol. The open reading frame (ORF) of AcCPR was cloned using the primers (Supplementary Table S1) that were designed based on the data of A. citricidus transcriptome. PCR was carried out using PrimeSTAR^® Max DNA Polymerase (TaKaRa Bio, Dalian, China). The PCR cycling conditions were as follows: 98°C for 3 min; 35 cycles (98°C for 15 s, 58°C for 15 s, 72°C for 2 min); and 72°C for 10 min. The amplified DNA was purified and ligated into the pGEM-T Easy Vector (Promega, Madison, WI, United States). Positive clones were verified by PCR using the universal primers M13F and M13R and sequenced (Invitrogen, Shanghai, China).

Cells were collected (7871× g for 6 min) after cultivation for 68 h. Cell pellets were then washed three times with distilled water and subsequently frozen at −80 °C for 2 h. The frozen cell pellets were ground in liquid nitrogen to fine powder then 100 mg powder was transferred into a clean 1.5 mL centrifuge tube. Subsequently 1 mL TRIzol (Life Technologies, Carlsbad, CA, USA) reagent was added into the tube and set in room temperature (RT) for 30 min, after that 0.2 mL chloroform was added and the mixture was vortexed for 30 s, rest in RT for 5 min then centrifuged at 17,709× g for 15 min. The upper layer was transferred to a new centrifuge tube and was added 0.5 mL isopropanol and placed in RT for 10 min. Total RNA precipitate was obtained after centrifugation at 17,709× g for 15 min at 4 °C, and then it was washed twice with 75% ethanol and dissolved with DNase/RNase free water. The integrity of RNA was checked by 1.5% (w/v) agarose gel electrophoresis. The quality and concentration of RNA were determined using a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Total RNA with 260/280 nm ratio of 1.9–2.2 and 260/230 nm ratio greater than 1.9 was used to synthesize cDNA with PrimerScript^TM RT reagent Kit (Takara, Japan) according to the manufacturer’s guidelines. The cDNA was stored at −20 °C for later use.

The total RNA was extracted from the gEECs using TRIzol reagent (TaKaRa, Tokyo, Japan), according to the manufacturer’s instructions. The RNA concentration and purity were measured using an Ultramicro spectrophotometer (ThermoScientifc, Waltham, MA, USA), following the PrimerScript^TMRT reagent kit (TaKaRa, Tokyo, Japan) instructions, and the RNA was converted into complementary DNA (cDNA). Total RNA = 0.4 μg, 5 ×PrimeScript RT Master Mix = 10 μL, and RNase Free ddH20 = up to 20 μL were used. The primer sequences are shown in Table S1. The amplification curve and melt curve of primers are shown in Figure S3. Real-time quantitative PCR (qPCR) was performed using SYBR Green Master Mix (Vazyme, Nanjing, China) in a Bio-Rad CFX96 (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s protocol. cDNA = 2 μL, 2 ×PrimeScript RT Master Mix = 10 μL, forward primer and reverse Primer (10 μM) = 0.8 μL, RNase Free ddH20 = up to 20 μL were used. The RT-PCR parameters were as follows: 37 °C for 15 min and 85 °C for 5 s. The qPCR parameters were as follows: 95 °C for 30 s, followed by 40 cycles each of 95 °C for 5 s and 60 °C for 20 s. The level of mRNA quantification was estimated with the 2^−∆∆ct method. The expression of mRNA was normalized to the GAPDH gene, which served as the control gene in all samples.

Quantitative real-time polymerase chain reaction (RT-qPCR) analysis was performed to validate the alteration in levels of DEGs revealed by RNA-seq analysis. Basically, total RNA was extracted from cells using HiPure Total RNA Mini Kit (Magen, Cat.No. R4111-02) and reverse transcribed into cDNA using the Primer ScriptTM RT Reagent Kit (TaKaRa, Cat.No. RR047Q). The prepared cDNA was then subjected to quantitative PCR analysis using TB Green® Premix Ex TaqTM (Tli RNaseH Plus) kit (TaKaRa, Cat.No. RR420Q) in a qTOWER 3G Cycler system (Analytik Jena AG, Germany). The sequences of the primers are presented in Table 1. The cycling condition was 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, and 60°C for 30 s. The fold induction was calculated as described previously [25 (link)].

Total RNAs in cells and tissues were extracted using Trizol reagent (Invitrogen, USA), according to the procedures as previously described [5 (link)]. Subsequently, 5 μg of total RNA was used for reverse-transcription into cDNA using RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). The expression of the target genes was detected by the PrimerScript^TM RT reagent Kit (Takara, Japan) on a 7500 Real-Time PCR System (Applied Biosystems, CA, USA). β-actin was used as the reference gene and the relative gene expression was calculated by the 2^−ΔΔCt method. The sequences of primers in this study are as follows: HMMR-AS1 (forward: 5’-CACAACTCCTGGTTCCTG-3’; reverse: 5’-GGATGGGTATTGGTCTGT-3’); miR-627-3p (forward: 5’-CACTCATCTTTTCTTTG-3’; reverse: 5’-GAGTCTCTTGAGAGTACAT-3’); β-actin (forward: 5’-CCACTGGCATCGTGATGGA-3’; reverse: 5’-CGCTCGGTGAGGATCTTCAT-3’).