Abi 7500 fast real time pcr system

RNA was extracted from cell lines with RNAiso plus (TaKaRa) and reversely transcribed into cDNA with the PrimeScript RT reagent Kit. SYBR Premix Ex Taq (TaKaRa) was used to quantify the transcribed cDNA on the ABI 7500 fast real-time PCR System (Carlsbad, U.S.A.). GAPDH mRNA were used as endogenous references to calculate the relative expression of associated genes with the 2^−ΔΔCt method. Three duplicate samples were analyzed in each group. All primers used are listed as follows: GAPDH: 5′-CGGATTTGGTCGTATTGGG-3′ (forward) and 5′-CTGGAAGATGGTGATGGGATT-3′ (reverse); MYB: 5′-GACCCGGGAAGAGGATGAAA-3′ (forward) and 5′-CTCCCCTTTAAGTGCTTGGC-3′ (reverse).

Total RNA was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. To remove the DNA, 2 µg of RNA was treated with DNase and then reverse-transcribed into cDNA using SuperScript First Strand cDNA System (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequently, with cDNA as the template, RT-qPCR was performed using an ABI 7500 Fast Real-Time PCR System with SYBR^® PremixExTaq™ kit (Takara Biotechnology Co., Ltd.). The qPCR thermocycling conditions were as follows: pre-denaturation at 95°C for 10 min; followed by 40 cycles of denaturation at 95°C for 15 sec, and annealing and extension at 60°C for 60 sec. The primers were designed and synthesized by BGI (Shenzhen, China). The expression levels of FGD5-AS1, miR-577, low-density lipoprotein receptor-related protein 6 (LRP6) and β-catenin were determined with 2^−∆∆Cq method (19 (link)). The levels of U6 were used to normalize the miR-577 expression, and the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to normalize the expression levels of lncRNA and mRNA. The sequences of the primers are listed in Table I.

Total RNAs were extracted from N2 and daf-2(e1370) worms on adult day 1 using TRIzol (Invitrogen), followed by removing contaminant DNA by DNase I treatment. The cDNA was synthesized by using a reverse transcription kit (Takara Bio). qPCR was carried out on an ABI 7500 Fast real-time PCR system using a Takara real-time PCR kit (SYBR Premix Ex TaqTM II). mRNA levels of pmp-3 and act-1 were used as the internal control.

Based on the transcriptome comparison between the three NILs and 93–11, several DEGs were selected for further confirmation by real-time quantitative PCR. Primary panicles of 3–6 cm length were used for total RNA extraction with an RNA extraction kit (RNAiso Plus, TaKaRa Bio, Inc.). Reverse transcription was performed using 6 μg RNA and 4 μg reverse transcriptase mix (PrimeScript® RT Master Mix Perfect Real Time, TaKaRa Bio) in a volume of 40 μl, according to the manufacturer’s protocol. Real-time PCR was carried out in a total volume of 25 μl containing 2 μl of cDNA, 0.2 mM gene-specific primers, 12.5 μl SYBR® Premix Ex Taq TM II, and 0.5 μl of Rox Reference Dye II (TaKaRa Bio), using an ABI 7500 Fast Real-Time PCR System according to the manufacturer’s instructions. The rice 18S rRNA gene was used as an internal control. Relative quantification of the transcript levels was performed using the 2^−ΔΔCT method [48 (link)].

To validate the transcriptome data, the relative expression of nine DEGs identified in the transcriptome analysis was assessed by performing qRT-PCR, using three biological and three technical replicates. We performed RNA extraction and cDNA synthesis using the TIANGEN Total RNA Extraction Kit (TIANGEN, Beijing, China) and PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan), respectively. The specific primers for selected DEGs were designed using the Primer premier 5.0 software (Premier Biosoft, Palo Alto, CA, USA) and are shown in (Supplementary Table S1). The actin gene was used as an internal control. qRT-PCR detection was performed on the ABI 7500 Fast Real-Time PCR System using the TaKaRa SYBR Green Master Mix Kit (TaKaRa, Beijing, China).

After the cotton plants produced buds, different tissues, including roots, stems, and leaves, were taken. Whole Arabidopsis thaliana seedlings were taken when they were mature enough for testing. The total RNA was extracted and then reverse transcribed into cDNA. The GbUBQ7 and AtACTIN8 genes were selected as internal reference genes, and Primer Premier 6.0 software was used to design specific quantitative primers (UBQ7F: 5′-GAAGGCATTCCACCTGACCAAC-3′; UBQ7R: 5′-CTTGACCTTCTTCTTCTTGTGCTTG-3′; ACTIN8F: 5′-ATCCTCCGTCTTGACCTTG-3′; ACTIN8R: 5′-TGTCCGTCAGGCAACTCAT-3′; GbD14F: 5′-TCCCAGGTTTCTCAATG-3′; GbD14R: 5′-CACGCAACACGGCACT-3′). The experiment was performed using an ABI 7500 Fast Real-Time PCR System with SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Dalian, China); the reaction procedure was 40 cycles of 95 °C for 20 s, 95 °C for 3 s, and then 60 °C for 30 s.