Miniopticon cfx 48 real time pcr detection system

Total microRNAs were extracted and purified from frozen mesenteric vessels by using miRNeasy Mini Kit (Qiagen, Germany). Reverse transcription of the extracted miRNAs was performed by the miScript Reverse Transcription Kit (Qiagen, Germany). cDNA was diluted 1:3 in RNase-free water and then qPCRs were performed in triplicate using the miScript SYBR-Green PCR kit (Qiagen, Germany) on the MiniOpticon CFX 48 real-time PCR Detection System (Bio-Rad, Hercules, CA, United States). MiScript Primer Assays specific for mmu-miR-214-3p (MIMAT0000661), mmu-SNORD6 and mmu-RNU6 were obtained from Qiagen. miR-214-3p expression was calculated using Ct method and normalized to the expression of two housekeeping, SNORD6 and RNU6.

Total microRNAs (miRNAs) were extracted and purified from frozen adipocytes by using miRNeasy Mini Kit (Qiagen, Germany). Reverse transcription of the extracted miRNAs was performed by the miScript Reverse Transcription Kit (Qiagen, Germany). cDNA was diluted 1:3 in RNase-free water and then qPCRs were performed in triplicate using the miScript SYBR-Green PCR kit (Qiagen, Germany) on the MiniOpticon CFX 48 real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) [56 (link),57 (link)]. MiScript Primer Assays specific for hsa-miR-126-3p, hsa-miR-223-3p, hsa-miR-124-3p, hsa-miR-155-5p and hsa-miR-132-3p and hsa-RNU6 were obtained from Qiagen, and the probe sequences are proprietary. miRNAs expression was calculated using the Ct method and normalized to the expression of housekeeping RNU6. Mature miRNA sequences and miRBase Accession are reported in Table 2.

Plasma, isolated from the whole blood as previously described (Section 2.4), was processed by using the miRNeasy Serum/Plasma Mini Kit (Qiagen, Hilden, Germany) to isolate total RNA, including microRNAs (miRNAs). Retrotranscription was carried out using the miRCURY LNA miRNA RT Kit (Qiagen, Hilden, Germany), and the obtained cDNA was diluted to 1 : 30, immediately before use. Real-time PCR was run on the MiniOpticon CFX 48 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the miRCURY LNA miRNA SYBR Green PCR and specific miRCURY LNA miRNA PCR Assay (Qiagen, Hilden, Germany), as previously reported [57 (link)]. The miRCURY Primer Assay specific for hsa-miR-195-5p (MIMAT0000461), hsa-miR-153-3p (MIMAT0000439), and hsa-miR-93-5p (MIMAT0000093) was purchased from Qiagen (Hilden, Germany).
The relative miRNA expression was calculated using the Ct method and normalized on miR-93-5p. Several pieces of evidence reported high stability of miR-93-5p in biofluids [58 (link)–61 (link)]; thus, miR-93-5p was suggested as a plasmatic reference gene in the manufacturer's handbook. According to this, the expression levels of miR-93-5p in plasma samples of our cohort showed comparable expression levels without significant difference among groups (data not shown) [49 (link)].

The miRNeasy Mini Kit (Qiagen, Hilden, Germany) was used for purification and extraction of total miRNAs. The extracted miRNAs were retro-transcribed by the miScript Reverse Transcription Kit (Qiagen, Germany) and the corresponding cDNA was diluted 1:3 in RNase-free water. The miScript SYBR-Green PCR kit (Qiagen, Germany) was used to perform qPCR experiments in triplicate. Signals were detected on the MiniOpticon CFX 48 real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). MiScript Primer Assays specific for hsa-miR-193a-3p (MIMAT0000459), hsa-let-7g-5p (MIMAT0000414) and hs-SNORD6 were obtained from Qiagen. miRNA expression was calculated using the Ct method and normalized to the expression of the SNORD6 housekeeping gene.

Total RNA from cells was extracted using the RNeasy Mini kit. Reverse-transcription and Real-time PCR were carried out one-step by using the QuantiNova SYBR Green RT-PCR Kit (Qiagen, Hilden, Germany) on 200 ng of each sample, following the manufacturer’s instructions. The sequences of forward and reverse primers are reported in Table 3. Signals were detected on the MiniOpticon CFX 48 real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). The mRNA expression was calculated using the 2^-δδCt method.

Total microRNAs were purified and extracted from cryo-preserved colon (20–50 mg) and epididymal fat (100mg) by using miRNeasy Mini Kit (Qiagen, Germany). Reverse transcription of the extracted miRNAs was performed by the miScript Reverse Transcription Kit (Qiagen, Germany). cDNA was diluted 1:10 in RNase-free water and then qPCRs were performed in triplicate using the miScript SYBR-Green PCR kit (Qiagen, Germany) on the MiniOpticon CFX 48 real-time PCR Detection System (Bio-Rad, Hercules, USA). MiScript Primer Assays specific for mmu-let-7f-5p (MIMAT0000525) and the housekeeping mmu-RNU6 were obtained from Qiagen. let-7f expression was calculated using Ct method and normalized to the expression of RNU6.

The miRNeasy Mini Kit (Qiagen, Hilden, Germany) was used for purification and extraction of miRNAs from exosomes isolated from cell culture conditioned supernatants or adipocytes. The retro-transcription of extracted miRNAs was performed by using the miScript Reverse Transcription Kit (Qiagen) [51 (link)]. The cDNA obtained was diluted 1:3 in RNase-free water from adipocytes, while the exosome-cDNA was used without dilution. The qPCR experiments were performed by miScript SYBR-Green PCR kit (Qiagen), as previously reported [52 (link)]. Signals were detected on the MiniOpticon CFX 48 real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). MiScript Primer Assays specific for hsa-miR-34a-5p (MIMAT0000255), hsa-miR-155-5p (MIMAT0000646) and hsa-let-7c-5p (MIMAT0000064) and hsa-SNORD6 were obtained from Qiagen. The expression of miRNAs was calculated using in the comparative cycle threshold (Ct) method and normalized to the expression of housekeeping genes SNORD6 for adipocyte-derived miRNAs, and Caenorhabditis elegans miR-39 (Cel-miR-39) for exosome-derived miRs (exo-miRs).