M mlv reverse transcriptase

Primers for the analysis of RNA expression were designed by Primer-BLAST (www.ncbi.nlm.nih.gov). Mixtures of 1 μg total RNA, 50 nM reverse primer, 2 units of RNase inhibitor (Toyobo, Osaka, Japan), 5 units of M-MLV reverse transcriptase (Toyobo) and 0.5 μM dNTP were used for each RT reaction. qRT-PCR was performed in 20-μL reactions with the Bio-Rad S1000 detection system (Toyobo).

Quantitative real-time RT-PCR (qRT-PCR) was used to verify RNA expression identified by Agilent Rat 8 × 60 K Microarray. The primers for the identified RNA were designed using Premier 5.0. Mixtures of 1 µg of total RNAs, 50 nM reverse primer, 5 units of M-MLV reverse transcriptase (Toyobo), 2 units of the RNAase inhibitor (Toyobo, Osaka, Japan) and 0.5 µM dNTP were incubated for 30 min at 16 °C, 30 min at 42 °C and 15 min at 70 °C. The reaction mixes were used as the templates for qRT-PCR using SYBR^® Green Real-time PCR Master Mix-Plus (Toyobo, Osaka, Japan) on the Applied Biosystems 7500 detection system. The PCR mixture including 1 µl RT product of total RNA, 10 µl SYBR-Green Real-time PCR Master Mix-plus, 2 µl plus solution, 2 µl each specific forward and reverse primers, and 3 µl DEPC water was made up the final volume to 20 µl. The reaction was performed at 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Each sample was ran in triplicate and actin was used as internal control. Melting curves were used for verifying the specificity of each PCR reaction. The cycle threshold (Ct) was the mean value of three Ct values. The relative expression level of RNA was analyzed by the 2^−∆∆Ct method.

According to the manufacturer’s instruction, total RNAs were extracted from the hLE cells using Trizol reagent (Invitrogen). cDNA was obtained by incubation of the mRNA with M-MLV reverse transcriptase (Toyobo, Osaka, Japan), oligo (dT) (Toyobo) and dNTPs (Toyobo) at 42 °C for 40 min in the buffer (Toyobo). After inactivation of the enzyme by incubation at 95 °C for 5 min, polymerase chain reaction (PCR) was carried. The primer sequences are as follow: SelR: 5′-ATGTCGTTCTGCAGCTTCTTC-3′ (forward) and 5′-CACACTTGCCACAGGACAC-3′ (reverse) [50 (link)]; GAPDH: 5′-CCATGTTCGTCATGGGTGTGAACCA-3′ (forward) and 5′-GCCAGTAGAGGCAGGGATGATGTTC-3′ (reverse) [51 (link)]; Real-time PCR was performed in DNA Engine Opticon 2 (MJ Research, Watertown, MA, USA). PCR conditions for SelR were 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min; for GAPDH, the conditions were referred [51 (link)].

Total RNA was extracted from EPCs using Trizol (Invitrogen, Carlsbad, CA, USA), and then cDNA was synthesized with oligo(dT) and MMLV reverse transcriptase (Toyobo, Japan). cDNA amplification and semi-quantitative PCR were performed using the following primer pairs.Slfn1 forward 5′-CCA GAT GTC TCT GTT GGG AA-3′ and Slfn1 reverse 5′-GCT AAG ACA TGA GGA GCT TG-3′[9] (link), Cyclin D1 forward 5′- TGC TTG GGA AGT TGT GTT GG-3′ and Cyclin D1 reverse 5′-AAT GCC ATC ACG GTC CCT AC-3′[15] (link), and rat β-actin forward 5′-TCA GGT CAT CAC TAT CGG CAA T-3′ and rat β-actin reverse 5′-AAA GAA AGG GTG TAA AAC GCA-3′. All primers, which were salt-free and of the highest purity, were synthesized by Sangon Biotech (Shanghai).

TriZol reagent (Invitrogen, USA) was used to isolate total RNA. To synthesize the first-strand cDNA, MMLV reverse transcriptase (Toyobo, Japan) was used as per manufacturer’s instructions. By using the leaf cDNA of LA1777, the full-length open reading frame (ORF) of ShCML44 was amplified. The sequence specific primer (ShCML44, Table S1) were designed according to S. lycopersicum full-length cDNA LOC101245247 (designated as SlCML44, GenBank accession no. XM_004237307.2). To clone the amplified PCR product, pEASY-blunt cloning vector (TransGen, China) was used and consequently the recombinant clones were sequenced to identify the correct sequence. For gene prediction online SoftBerry tool (http://linux1.softberry.com/berry.phtml) and for sequence alignment ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) were used.

To identify and validate the differentially expressed miRNAs in the photosynthesis pathway, six miRNAs with high expression levels (at least 1000) in all libraries were selected from conserved families (miR171f, miR319, MiR156a, and miR159), and non-conserved (MIR305714, MIR46018, and MIR2836) and amplified using real-time qPCR to examine their expression. The forward miRNAs primers were designed based on the full miRNAs sequence, while the reverse primer was the universal reverse primer (5’GTGCAGGGTCCGAGGT3’). The stem-loop primer, used for miRNA cDNA synthesis, was designed according to Chen et al. (2005) (link). The 18S rRNA was taken as the reference gene. Similarly, the expression profiles of the respective miRNA target genes were assayed by real-time qPCR. In summary, a 10-µg aliquot of RNA was used for the RT reaction followed by the addition of oligo(dT) primer. The RT reaction was performed by MMLV reverse transcriptase (Toyobo, Osaka, Japan) according to the supplier’s manual. For real-time qPCR, ACTIN for bermudagrass was used as a housekeeping gene as the internal control. To calculate the relative expression, the 2^-ΔΔCt method was used and Student’s t-test was performed to compare differences in expression profile. The means were considered significantly different when P ≤ 0.05.