Gf 1 total rna extraction kit

RT-PCR: to detect the expression of fawrky1 gene in the elicited strawberry tissues with different elicitors, total RNA of strawberry calli (Camarosa) obtained from different elicitation treatments was extracted as recommended by GF-1 total RNA extraction kit (Vivantis). The full length of the fawrky1 gene was isolated using specific primers WR-F “5′ATGGATACCTACCCAGCATTC3” and WR-R “5′TCACAAAGAAGTGTAGATTTGCAT3”, (EU727547). RT-PCR and the amplification reactions were performed by following the instruction procedure of the two-steps-RT-PCR kit (Vivantis), cDNA was produced with 2 µg of total RNA of strawberry.
Sequence and alignment: the obtained fragments of the different treatments were then purified and cloned into PGEM–T Easy vector and transferred to the cell E. coli of strain DH5 α. Screening of the transformed colonies was performed with EcoR1 digestion to choose the right colony carrying the gene of interest. The isolated DNA from each sample was then sequenced and all sequences using sp6 universal primer. Alignment was performed using http://www.ncbi.nlm.nih.gov/.

Total RNA was isolated from cells using a GF-1 Total RNA Extraction Kit (Vivantis), according to instructions from the manufacturer. The complementary DNA (cDNA) was converted from RNA templates using a TaqMan Reverse Transcription Kit (Thermo Scientific). qPCR (SYBR Master Mix, Biotechrabbit) for TRF1, TRF2, and POT1 was performed. The qPCR conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 20 s. The primers (36 (link)) were TRF1: (F) 5′-GCTGTTTGTATGGAAAATGGC-3′, (R) 5′-CCGCTGCCTTCATTAGAAAG-3′, TRF2: (F) 5′-GACCTTCCAGCAGAAGATGCT-3′, (R) 5′-GTTGGAGGATTCCGTAGCTG-3′, POT1: (F) 5′-TCAGATGTTATCTGTCAATCAGAACCT-3′, (R) 5′-TGTTGACATCTTTCTACCTCGTATAATGA-3′, and GAPDH: (F) 5′-AACGTGTCAGTGGTGGACCTG-3′, (R) 5′- AGTGGGTGTCGCTGTTGAAGT-3′.

Total RNA was extracted from api-AuNPs-treated and untreated KKU-M055 cells using GF-1 total RNA extraction kit (Vivantis Technologies, Selangor, Malaysia) according to the manufacturer's instructions. The first strand complementary DNA (cDNA) was synthesized by reverse transcription with oligo d(T) primers using the Improm II™ reverse transcriptase system (Promega). RT-PCR was performed to detect the expression level of apoptotic genes (Bax, Bid, and Caspase 3), pro-survival gene (Bcl-2) and reference gene (Actin), using a Toptaq Master Mix Kit (Qiagen, Hilden, Germany). The PCR reaction of the above genes was carried out in a T100 Thermal Cycler PCR system (Bio-Rad Laboratories, Hercules, CA, USA) using the primer sequences as shown in Table 1.Table 1

Primer sequences for RT-PCR.

Table 1

Genes	Forward primer sequences (5′–3′)	Reverse primer sequences (5′–3′)	Product size (bp)
Bax	CATCCAGGATCGAGCAGG	CATGTCAGCTGCCACTCGG	208
Bid	CAAGAAGGTGGCCAGTCACAC	GCTCCGTCTACTCTGGAAGC	199
Caspase 3	GTTGATGATGACATGGCGTG	GTTGCCACCTTTCGGTTAA	203
Bcl-2	GACTTCGCCGAGATGTCCAG	GTTCAGGTACTCAGTCATCCA	216
Actin	CTCCTCCCTGGAGAAGAGCT	GCAATGCCAGGGTACATGGT	231