Prism 7500 real time pcr system

Quantitative RT-PCR analyzed the expression profiles of candidate genes in transgenic cotton plants and wild type subjected to salt stress. Quantitative RT-PCR was performed in triplicate with an ABI PRISM® 7500 Real-Time PCR System using the SYBR® Premix Ex Taq™ Kit (TaKaRa, DRR041A), according to the manufacturer’s instruction. The gene-specific primers were shown in Additional file 4 Table S4. The GhUBQ7 transcript was used as a normalization control to quantify relative levels. Relative expression levels were calculated using the 2^-∆∆t method [91 (link), 92 (link)]. Standard deviation was calculated from three biological replicates.

The TRIzol Reagent (Invitrogen) was used for extract total RNA from tissues and cells. RNA was reversely transcribed into cDNA using a miScript II RT kit (TaKaRa, Biotechnology Ltd., Dalian, China) and then the real-time qPCR was performed using a Premix Ex TaqTM II (TaKaRa) on an ABI PRISM 7500 real-time PCR System. Relative gene expression was determined by the 2^-ΔΔCt method. The primers are listed in Table 1, in which U6 and GAPDH were set as the internal references for miRNA and mRNAs, respectively.
Table 1

Primer sequences for RT-qPCR

Gene	Primer sequence (5′-3′)
miR-545	F: CGACAAGGGTCAGCAAACATT
	R: GCAGGGTCCGAGGTATTC
Bax	F: TCAGGATGCGTCCACCAAGAAG
	R: TGTGTCCACGGCGGCAATCATC
Bcl-2	F: ATCGCCCTGTGGAGAACTACTGAGT
	R: GCCAGGAGAAATCAAACAGAGGC
KDM4B	F: GCCGAGAGGAAGTTCAACGCAG
	R: TGCCTCCTTCTCAGAGTGTGTAGG
PLK1	F: GCACAGTGTCAATGCCTCCAAG
	R: GCCGTACTTGTCCGAATAGTCC
U6	F: CTCGCTTCGGCAGCACA
	R: AACGCTTCACGCATTTGC
GAPDH	F: GTCTCCTCTGACTTCAACAGCG
	R: ACCACCCTGTTGCTGTAGCCAA

Note: RT-qPCR reverse transcription quantitative polymerase chain reaction, Bcl-2 B-cell lymphoma-2, Bax Bcl-2-associated X, KDM4B Lysine (K)-specific demethylase 4B, PLK1 Polo-like kinase 1, GAPDH glyceraldehyde-3-phosphate dehydrogenase, F forward, R reverse