Taqman mir reverse transcription kit

Using Trizol reagent (Life Technologies, New York, USA), total RNA was isolated from tissues or cells according to standard instructions. Then synthesis of the first strand of complementary DNA (cDNA) was via either TaqMan Reverse Transcription Kit (for circ-UBR1 and SSFA2) or TaqMan miR Reverse Transcription Kit (for miR-545-5p) (Applied Biosystems, Foster City, CA, USA). SYBR Select Master Mix (Roche, Basel, Switzerland) was applied to perform qRT-PCR on CFX Connect Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The reaction conditions were 95°C, 2 min; 95°C, 30 s, 60°C, 30 s and 72°C, 30 s, and 40 cycles [23 (link)]. Normalization of relative expression was to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6, and calculation of the expression was via 2^−ΔΔCt. Each experiment was repeated three times. Primer sequences are clarified in Table 1.Table 1.

Primers for RT-qPCR

Genes	Primer sequences (5ʹ – 3ʹ) PCR product
Circ-UBR1	F: TCTGTGCAATACTGTGATCCCC 132bp
	R: GGAGCTTTTTGAAGCTGTTGCT
MiR-545-5p	F: AGCGCGTCAGTAAATGTTTATT 110bp
	R: GTTGTTGGTTGGTTGGTTGT
SSFA2	F: TGGCAAGAAAGGCCCCTGTG 100bp
	R: GGAGCAGCAGCAGGATCAGG
U6	F: CTCGCTTCGGCAGCACA 108bp
	R: AACGCTTCACGAATTTGCGT
GAPDH	F: CACCCACTCCTCCACCTTTG 95bp
	R: CCACCACCCTGTTGCTGTAG

RNA was isolated via QIAzol extraction as described above from 293T cells, HeLa cells, six B-cell lines homozygous for rs59097151A or rs59097151G, two B-cell lines heterozygous for rs59097151, and fresh PBMC samples from each of three RDP donors. The expression of miRNA species was determined via TaqMan miR Assays for miR-4486 (465336_mat), miR-423 (000576), and RNU48 (001006) with the TaqMan miR Reverse Transcription Kit and the TaqMan Fast Advanced Master Mix (Applied Biosystems). hsa-miR-423 and RNU48, a small nuclear RNA, served as controls that were stably expressed across the cell types examined (SI Appendix, Fig. S4A). By using the 2^-ΔΔCt method, the qPCR results were analyzed for miR-4486 expression with RNU48 as a reference for expression and miR-423 as the endogenous control.

Total RNA was isolated from cells using the PureLink RNA Mini Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. The mRNA reverse transcription was conducted using the iScript cDNA synthesis kit (Bio‐Rad Laboratories, Hercules, CA, USA), or the mature miR reverse transcription was accomplished using the TaqMan miR Reverse Transcription Kit (Applied Biosystems, Inc., Foster City, CA, USA). ABI StepOne Real‐Time PCR System (Applied Biosystems) and SYBR Green Mix (Bio‐Rad) were utilized for mRNA expression, or TaqMan Universal Fast PCR Master Mix for miRNA expression. Relative mRNA and miRNA expression was examined using the 2^−∆∆Cq method and normalized to the levels of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and U6, respectively. The primers were synthesized by Sangon (Shanghai, China): miR‐125a, forward 5′‐CGGTCCCTGAGACCCTTTAAC‐3′, reverse 5′‐GTGCAGGGTCCGAGGT‐3′; HDAC2, forward 5′‐TAAATCCAAGGACAACAGTGG‐3′, reverse 5′‐GGTGAGACTGTCAAATTCAGG‐3′; PHOX2B forward 5′‐AGTGGCTTCCAGTATAACCCG‐3′, reverse 5′‐GGTCCGTGAAGAGTTTGTAAGG‐3′; MYCN forward 5′‐ACCCGGACGAAGATGACTTCT‐3′, reverse 5′‐CAGCTCGTTCTCAAGCAGCAT‐3′; U6 forward 5′‐CTCGCTTCGGCAGCACA‐3′, reverse 5′‐AACGCTTCACGAATTTGCGT‐3′; and GAPDH forward 5′‐GATTCCACCCATGGCHDAC2TTC‐3′, reverse 5′‐AGCATCGCCCCACTTGATT‐3′.

Total RNA was isolated from the cells using the PureLink™ RNA Mini Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription reactions were performed using iScript cDNA synthesis kit (Bio‐Rad) for mRNA, or TaqMan miR Reverse Transcription kit (Applied Biosystems) for mature miR as described earlier (Challagundla et al., 2015). A qRT‐PCR assay was performed using an ABI StepOne real‐time PCR system (Applied Biosystems) with SYBR green mix (Bio‐Rad) for mRNA expression or TaqMan Universal Fast PCR master mix for miRNA, as described earlier (Challagundla et al., 2011, 2015; Dai et al., 2012; Sun et al., 2012). All reactions were carried out in triplicate. Relative gene or miRNA expression levels were normalized with GAPDH mRNA or U6 small nucleolar RNA (snoU6) and were calculated using the ΔCτ method. The qRT‐PCR primer sequences for MYCN and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) were as follows: (MYCN) Forward: 5′‐ACCCGGACGAAGATGACTTCT‐3′, Reverse: 5′‐CAGCTCGTTCTCAAGCAGCAT‐3′; (GAPDH) Forward: 5′‐GATTCCACCCATGGCAAATTC‐3′, Reverse: 5′‐AGCATCGCCCCACTTGATT‐3′ (Spandidos et al., 2010; Wang and Seed, 2003).