M mulv reverse transcriptase

Total RNA extracted from human tissues, U87, LN229, and U251 cells using Trizol reagent (Life Technologies) was reverse-transcribed to the first-strand cDNA in a 20-µl reaction volume containing 5 µg RNA, random primer, dNTP, RNAase inhibitor, and M-MuLV reverse transcriptase (Promega) for 25°C, 10 min, then 42°C, 1.5 h, and 72°C, 10 min. qPCR was performed with SYBR Premix ExTaq Kit (Genestar) and analyzed on Step One plus (ABI). The qPCR conditions were as follows: 95°C, 10 min; 95°C, 10 sec, 60°C, 15 sec, 72°C, 20 sec, and additional 39 cycles. Quantification was done by using the comparative Ct (ΔΔCt) method. The primers used for qPCR are listed in Supplementary Table S1.

Total RNA was isolated from the prostate tissues of each mouse using Trizol (Invitrogen, CA, USA), according to the manufacturer's instructions. An aliquot of total RNA was reverse transcribed using MMuLV reverse transcriptase and Taq DNA polymerase (Promega, Madison, WI, USA), respectively. Primers for VEGFA, IL6, IL-1ß, TNF-α, and COX2 were designed in reverse transcription (RT- PCR) according to previous studies [18 (link), 19 (link)].

After RNAs qualification, sequencing libraries were constructed using Multiplex Small RNA Library Prep Set for Illumina (NEB, USA) following manufacturer’s recommendations. Briefly, 3ʹ SR adaptor was directly, and specifically ligated to 3ʹ end of miRNA, siRNA and piRNA. Then first strand cDNA was synthesized using M-MuLV Reverse Transcriptase (Promega, Madison, USA). The library preparations were sequenced on an Illumina Hiseq 2500/2000 platform and 50 bp single-end reads were generated. After quality control of raw data, miRNA expression levels were counted and normalized by transcript per million (TPM).

Dh404-treated normal and diabetic HAECs were stimulated with TNF-α (1 ng/ml) for 2 h. Following which, cells were washed twice with ice-cold PBS and lysed with RNA lysis buffer containing DNAse enzyme. After an incubation period of 7 min at room temperature with gentle agitation, a stop solution was added and DNAase-treated RNA was collected and its concentration was determined using the nanodrop spectrophotometer at 260 and 280 nm. Following which, DNA-free RNA (3 µg) was reverse-transcribed into cDNA using the Superscript First Strand System. 50 ng/µl of random primers were first added to RNA samples and incubated at 70 °C for 5 min, followed by immediate placement on ice. Meanwhile, a master mix was made from 4 μL of 5X first strand buffer, 2 μL of 10 mM dNTPs, 2 μL of 0.1 M dithiothreitol, 0.1 μL of RNase inhibitor (Promega; 20 U/μL) and 1 μL of M-MuLV Reverse Transcriptase (200 U/μL). The master mix was then added to the RNA mixture and incubated at room temperature (25 °C) for 10 min and subsequently incubated at 37 °C for 60 min and at 70 °C for 10 min to complete the reverse transcription. The mixture was then pulse centrifuged to pellet any condensation and the cDNA was then stored at −20 °C until further use.

To determine the co-transcription of the csa1, cas1, cas2 and cas4 genes, three gene-specific reverse primers (cas1qR, cas2qR and cas4qR; Supplementary Table S1, Figure 1) were used to generate the first-strand cDNA from the total RNA sample. Three forward primers (csa1qF, cas1qF and cas2qF; Supplementary Table S1, Figure 1) in combination with the three reverse primers listed above were used to amplify the first-stand cDNAs (Figure 1). The PCR products were separated on a 1% agarose gel. For real-time reverse transcription-PCR (RT-qPCR), first-strand cDNA was synthesized using M-MuLV reverse transcriptase (Promega, Madison, WI, USA) and the specific reverse primers (csa1qR, cas1qR, cas2qR and cas4qR; Supplementary Table S1). Each real-time quantification reaction was carried out as described above for the ChIP-qPCR assay using the first-strand cDNAs as template and each forward primer (csa1qF, cas1qF, cas2qF, cas4qF or csa3aqF) in combination with the reverse primers listed above along with csa3aqR. The transcripts of the albA gene were used as the control (45 (link)) and the cycle threshold (Ct) values of the control transcript albA were used to normalize the Ct values of the csa1, cas1, cas2, cas4 and csa3a transcripts.