Primer express software 3

RNA isolation from pericarp of the fruits was conducted using the
method described by Moore et al. [78 (link)]. The extracted RNA was treated with DNase I
(Promega [79 ]) and reverse
transcribed with Moloney murine leukemia virus (M-MLV) reverse transcriptase
(Promega).
Quantitative real-time PCR was performed with SYBR Green PCR Master
Mix (Applied Biosystems) using the StepOne Plus Real-Time PCR System (Applied
Biosystems). Gene-specific primers (Additional file 12) were designed with the Primer Express software 3.0 (Applied
Biosystems). The following PCR program was used: 95°C for 10 min, followed by 40
cycles of 95°C for 15 s and 60°C for 30 s. Relative quantification of specific
mRNA levels were measured using the cycle threshold (Ct)
2^(-ΔCt) method. Expression values were normalized
using actin (SGN-U580609). Three independent
biological replicates were analyzed for each sample.

Total RNA was isolated from 5 × 10⁸ yeast cells of each sample using TRIzol (Tiangen Biotech, Beijing, China). Genomic DNA elimination and first-strand cDNAs generation were performed using PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time, TaKaRa Code. DRR047, Dalian, China). The RT-qPCR reactions were carried out in a total volume of 20 μL, containing 10 μL of SYBR Premix Ex Taq^TM (TaKaRa Code. RR820), 0.4 μL of each primer (10 μM), 0.4 μL of ROX Reference Dye, 2 μL of cDNA, and 6.8 μL of RNase-free water. An ABI Step One Plus Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA) was used. The primers used for amplification were designed using Primer Express software 3.0 (Applied Biosystems), and the sequences were listed in Table 3. Transcript levels were normalized against the 5.8S rRNA (GenBank accession number AB568347.1) according to Li et al. [40 (link)] and relative expression levels were calculated using the 2^−ΔΔCT method [41 (link)].

qPCR was performed in the presence of SYBR Green fluorescent dye using a StepOne Real-time PCR system (Applied Biosystems, CA, USA). Briefly, the reaction mixture consisted of reverse transcribed cDNA, 2X POWER SYBR Green master mix (Applied Biosystem), and 10 μM of forward and reverse primers. The primer sequences for MGMT, p16, and β-Actin gene were selected from published articles [38 (link)–40 (link)] and synthesized by Eurofins MWG Operon, India. Primer sequences were cross-checked by Primer Express software 3.0 (Applied Biosystems, USA) and Blast sequence analysis (National Centre for Biotechnology Information, USA). Relative gene expression was determined by the 2^−ΔΔCt method using beta-actin as an endogenous control [37 (link)]. A negative control without template was run in parallel to assess the overall specificity of the reaction. All reactions were run in triplicates.

Total RNA was isolated from colonoids or cultured cells using the RNeasy Mini Kit (Qiagen, Valencia, CA) and reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real time PCR used the ABI Step OnePlus real-time PCR system and Power SYBR green PCR master mix (Applied Biosystems). Primers were designed by Primer Express software 3.0 (Applied Biosystems) based on nucleotide sequences from the National Center for Biotechnology Information data bank (Suppl. Table S3). For each sample, glyceraldehyde-3-phosphate dehydrogenase was used as the internal reference. All assays were performed in triplicate and fold changes were calculated using the ΔΔCT method.

Total RNA from mature leaves, flower buds, and fruits of litchi cultivars were extracted using the RNAout 2.0 kit (Tiandz, China). Total RNA was reverse transcribed into cDNA with the aid of the PrimeScript^TM RT reagent kit (Takara, Japan). Transcript levels of the LcFLS gene were analyzed using qRT-PCR with a CFX Connect^TM Real-Time PCR Detection System (Bio-Rad, United States) and SYBR^® Premix Ex Taq^TM II (Takara, Japan) according to the manufacturer’s instructions. Specific qRT-PCR primers were designed with Primer Express Software 3.0 (Applied Biosystems, United States) (Table 2). Gene expression values were calculated using the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)) and normalized using the LcActin gene (GenBank accession number HQ615689) as a reference. All reactions were performed in triplicate (with three biological replicates).

Isolation of total RNA and cDNA synthesis of NIH3T3 cells was performed as described in.⁴ (link) mHif1- α forward and reverse primers were designed via Primer Express Software 3.0 (Applied Biosystems, USA): mHif1- α: accession numberNM_001313919.1 (forward (f): 5ʹ-GAG TCT GAA GTT TTT TAT GAG CTT GCT-3ʹ, reverse (r): 5ʹ-GGT GAG CCT CAT AAC AGA AGC TTT-3ʹ). Primer concentrations were optimized through a primer matrix testing the combinations of three different primer concentrations each, followed by dissociation curves to test for the product yield and the presence of unwanted products such as primer dimers. For absolute quantification of mHif-1α mRNA calibration curves were generated as outlined in.⁴ (link) cDNA samples were measured in a QuantStudio™3 Real-Time PCR System using the Power SYBR® Green Master Mix (Thermo Fisher Scientific, UK). CT-values were calculated reciprocally to log10, according to the respective gene calibration curves, to achieve the absolute copy numbers. Absolute copy numbers were finally normalized to 10 ng of total RNA.
mHif1- α: accession numberNM_001313919.1 (forward (f): 5ʹ-GAG TCT GAA GTT TTT TAT GAG CTT GCT-3ʹ, reverse (r): 5ʹ-GGT GAG CCT CAT AAC AGA AGC TTT-3ʹ);

RNA was isolated from the pericarp using the method of Moore et al. [69 (link)]. The extracted RNA was treated with DNase I (Promega) and reverse transcribed using an oligo (dT)₁₈ primer with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega) to synthesize cDNA. Quantitative RT-PCR was carried out with the SYBR Green PCR Master Mix (Applied Biosystems) using the StepOne Plus Real-Time PCR System (Applied Biosystems). Gene-specific primers (Additional file 12: Table S11) were designed with the help of the Primer Express software 3.0 (Applied Biosystems). The following program was applied for PCR amplification in a volume of 20 μL: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Relative quantification of specific mRNA levels was performed using the cycle threshold (Ct) 2^(−ΔCt) method [70 (link)]. Expression values were normalized using ACTIN (SGN-U580609). Each experiment had three biological repeats, each with three technical replicates.