Methodology
After obtaining permission from the local Biodiversity Board, samples were collected from table fields of Pampore, Jammu, and Kashmir and the specimen was submitted in KASH herbarium, University of Kashmir under voucher specimen No. 4341-KASH-Herbarium. All the experiments were carried out following international guidelines.
Reagents
Trizol Invitrogen, Extraction buffer containing (2%(w/v) SDS; 0.05 M, Tris–HCl (pH = 7.5); 0.25 M EDTA(sigma); and Polyvinylpyrrolidone (PVP) (4%); NaCl, 20 mM) was taken from the protocol proposed by Chan et al. [14] and Liu et al. [15] with slight modifications. Aside from that, phenol: Chloroform: Isoamyl alcohol (24:1v/v) (PCI 1:1 v/v), and 100% acetone were used to remove secondary metabolites that are highly soluble in acetone (Merck).
RNA extraction
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1.
50 mg of tissue was crushed to fine powder in liquid nitrogen and transferred into 1.5 ml RNAse-free tubes.
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2.
The samples was washed twice with 1 ml of 100% acetone, and centrifuged at 12,000×g for 5 min.
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3.
500 μl of extraction buffer and 200 μl of β-mercaptoethanol (Sigma Aldrich) were applied to the pellet obtained. After vigorous mixing, the tubes were incubated at 25 °C for 15 min and centrifuged at 4 °C for 10 min at 12,000×g.
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4.
The supernatant (~ 300 μl) was decanted into a 1.5 ml tube and 300 µl of (1:1) Phenol: chloroform Isoamyl alcohol (24:1) was added to it and mixed thoroughly for about 2 min.
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5.
The tubes were centrifuged at 12,000×g for 10 min at 4 °C and 1/10 volumes of 3 M sodium acetate (PH 5.2) and 2.5 volumes of 100 percent ethanol were applied to the supernatant, and incubated for 1 h at 4 °C.
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6.
After centrifugation at 4 °C for 10 min at 12,000×g, the pellet was dissolved in 50 µl of Milli-Q.
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7.
To remove any DNA contamination, the samples were treated with DNase I (Invitrogen).
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8.
300 µl of milli-Q and 200 µl (1:1) phenol: Chloroform isoamyl alcohol (24:1) was added to the tubes and mixed gently.
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9.
After centrifugation at 4 °C for 5 min at 12,000×g, the upper aqueous layer (~ 300 μl) was transferred into a 1.5 ml tubes.
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10.
To recover the nucleic acids, 1/10 of 3 M sodium acetate (PH 5.2) and 2.5 volumes of 100% ethanol (Merck) was added to the tubes and incubated at 4 °C for 1 h.
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11.
The samples were centrifuged at 4 °C for 10 min at 12,000×g and the pellet was dissolved in 200 μl of RNase-free water.
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12.
500 μl of 10 M LiCl was added to the tubes and incubated on ice for 1 h followed by centrifugation at 4 °C for 10 min at 12,000×g.
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13.
The pellet was washed twice with 500 μl of 70% ethanol and allowed to dry in a speed vac for 2–5 min and re-suspended in 100 μl RNase-free water (Qiagen).
Analysis of RNA
RNA isolated from different tissue of Crocus sativus was analyzed on 1.5% (w/v) agarose gel, and its quality were assessed by Nanodrop & Agilent TapeStation.
cDNA synthesis and PCR
The cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo scientific). For miRNA-specific cDNA synthesis, 2 µg of total RNA was used as a template in a reaction comprising Reverse transcriptase, reverse tubulin primer, and specific miRNA stem-loop RT primers that bind to the 3′ end of a mature miRNA, reverse transcribing the miRNA to the corresponding cDNA. For each PCR reaction, 1 μl of cDNA was added to the reaction mixture containing 5 μl of 10 × PCR buffer, 0.5 μl 10 mM dNTP Mix, 0.5 μl of 10 μM forward, 0.5 μl of Reverse Primer, and 15.3 μl of H2O2. The PCR program included: initial denaturation at 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 40 s last extension at 72 °C for 10 min.
Small RNA library preparation and qPCR analysis
NEBNext Multiplex Small RNA Library Prep kit New England (Biolabs) was used to prepare small RNA libraries. To evaluate the miRNA expression profile, specific cDNA was used to amplify the tubulin and candidate miRNA using primers specific to stem-loop RT primer and mature miRNA. (Additional file 1: Table S1). Tubulin was used as an internal control to normalize the expression of miRNAs. The primer efficiency was calculated using ten-fold serial dilution. For each qPCR reaction, 1 μl of cDNA diluted by a factor of 10 was mixed in a 0.2 ml RNase free tube containing 6 μl of 2X Syber green, 0.3 μl each of forward and universal reverse primer, and 4.4 ml Milli-Q water. The reaction mixture was run in lightCycler480-II (Roach) and the fold chnage was calculated using 2−∆∆CT method [16].