Abi 7900 detection system

RT-qPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) on the ABI 7900 detection system (Applied Biosystems). Relative expression values were determined using the standard curve method. RT-qPCR was performed on bulk tuft cells and non-tuft epithelial cells from CD-1 mice with pancreatitis (100 cells per group) after the amplification step of the SmartSeq2 protocol (Picelli et al., 2014 (link)). Results were normalized to the housekeeping gene Rplp0. Primer sequences can be found in Supplementary Table S2.

Total RNA from articular cartilage and chondrocyte cultures was isolated using QIAzol reagent (Qiagene). One μg total RNA was subjected to reversed transcription. Amplification was performed using a ABI 7900 Detection System (Applied Biosystems), with 2 × TaqMan^® Universal PCR Master Mix. Primers for Atg4, Atg12, p62, Beclin, collagen II, aggrecan, SOX9, IL-1β, CXCL9, and calibrator gene 18S rRNA (Supplementary Table 1). Changes in mRNA expressions were calculated as 2^−ΔΔCt, where ΔΔCt = ΔCt_ACLT – ΔCt_sham and ΔCt = Ct_gene − Ct_18S.

Total RNA was extracted from WT and miR-29aTg osteoblasts using TRI Reagent™ Solution (Thermo Fisher Scientific Inc., Waltham, MA, USA). High-Capacity cDNA Reverse Transcription Kits (Thermo Fisher Scientific Inc., Waltham, MA, USA) were utilized to reversely transcribe 1 μg of total RNA. PCR was investigated using primers (Supplementary Table S1), 2× TaqMan^® Universal PCR Master Mix with ABI 7900 Detection System (Applied Biosystems, Foster City, CA, USA), and the threshold value for amplification reaction was probed. Equation 2^−ΔΔCt was used to calculate the fold change of mRNA expression.

Total microRNA in cultured cervical cancer cell lines was isolated using MicroRNA isolation kits (BioChain Institute, Inc., Hayward, CA, USA) according to the manufacturer’s instructions [63 (link)]. Aliquots of total microRNA (equivalent to 100 ng total RNA) were combined with a reverse transcription (RT) mixture (Ambion, Inc., Austin, TX, USA) and reverse-transcribed into complementary deoxyribonucleic acid (cDNA). All of the templates were mixed with polymerase chain reaction (PCR) mixtures and double that amount of TaqMan^® Universal PCR Master Mixture, and then we performed a PCR amplification with an ABI 7900 Detection System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Specific RT and PCR primers for endogenous control housekeeping gene U6 small nuclear RNA (U6) or miR-128 were purchased from Ambion, Inc. The fold change was estimated as 2−ΔΔCt, where ΔΔCt = ΔCttreatment − ΔCtsham control and ΔCt = Cttarget gene − CtU6, as we previously reported [63 (link)].

Total RNA was isolated from cardiomyocytes utilizing TRIzol reagent (Thermo Fisher Scientific, Waltham, MA), followed by reverse transcription with the application of a reverse transcription system (Thermo Fisher Scientific). qRT-PCR was conducted on ABI 7900 Detection System (Applied Biosystems, Foster City, CA) by use of the SYBR-Green PCR Master Mix kit (Takara, Shiga, Japan). Relative expression of genes, normalized to GAPDH or U6, was calculated via the 2^−ΔΔCt approach.

Articular tissues from injured rat joints were dissected using a surgical microscope. Specimens were homogenized using a Precellys 24^® homogenizer and a liquid nitrogen cooling system (Bertin Technologies, Montigny-le-Bretonneuz, France). Total microRNA was then extracted from the homogenates using the Biochain^® MicroRNA Isolation Kits (Biochain Institute Inc, Newark, CA). MicroRNA expression was detected using Megaplex^TM Pool arrays (Applied Biosystems Inc, Foster City, CA), according to the manufacturer’s instructions. Next, 1 μg of total microRNA was reverse transcribed by MultiScribe^® reverse transcriptase, 10×Megaplex^TM RT primers, dNTP and 10×RT Buffer and templates and further incubated with 2×TaqMan^® PreAmp Master Mix and 10×Megaplex^TM PreAmp Primers using an ABI 7900 Detection System (Applied Biosystems Inc, Foster City, CA). The start of the logarithmic amplification of the PCR reactions was computed automatically and interpreted as cycle threshold (Ct). Relative expression was calculated according to Eq. 2^−ΔΔCt, where ΔΔCt = ΔCt_ACLT −ΔCt_sham and ΔCt = Ct_microRNA − Ct_U6, was adapted to quantify changes in microRNA expression in the ACLT group. Potential microRNA candidates were selected based on an at least fivefold change cutoff in relative expression.