Three constructs from each time point were dissolved in 100 mM sodium citrate, containing 0.08 U∙μL−1 Proteinase K and 1.0 U∙μL−1 RNAse Inhibitor (A&A Biotechnology), while shaking for 5 minutes at 37°C, followed by ribonucleic acid (RNA) isolation with TriReagent (Sigma- Aldrich). Chloroform was then added, and the probes were centrifuged at 12,000 RCF for 15 minutes at 4°C. The supernatant was collected and mixed with a 1:1 volume of cold 99% ethanol. The solution was then transferred to the columns from RNeasy Mini Kit. The isolation steps were performed according to the RNeasy Mini Kit manual. The RNA concentration was measured using the Qubit 4 Fluorometer. For reverse transcription polymerase chain reaction (RT-PCR), TranScriba Kit (A&A Biotechnology) was used with random hexamer primers and 300 ng of total RNA. The following genes for real-time PCR were selected: COL1A1, COL2A1, COL10A1, SOX9, and RUNX2, with GAPDH as the housekeeping gene. The designed starters are shown in Table 2 . The QuantStudio 6k Flex Real-Time PCR System (Applied Biosystems) with 1 μL of complementary deoxyribonucleic acid (cDNA) and Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) was used to evaluate the expression of these genes. Primers were used at a final concentration of 0.5 μM. The gene expression results were tested with the twoway analysis of variance (ANOVA).
Rnase inhibitor
Manufactured by A&A Biotechnology
Sourced in Poland
The RNAse Inhibitor is a laboratory reagent used to prevent the degradation of RNA by inhibiting the activity of RNAse enzymes. It maintains the integrity of RNA samples during various molecular biology procedures.
Automatically generated - may contain errors
Lab products found in correlation
Maxima sybr green rox qpcr master mix, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Merck Group (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Transcriba kit, by A&A Biotechnology (1 mentions)
Quantstudio 6k flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantstudio 7 flex, by Thermo Fisher Scientific (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Glycoblue coprecipitant, by Thermo Fisher Scientific (1 mentions)
Rna extracol, by EURx (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
4 protocols using rnase inhibitor
1
RNA Extraction and RT-PCR Analysis of Chondrogenic Markers
2
Gene Expression Analysis Using qPCR
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Three constructs from each time point were dissolved in 100 mM sodium citrate containing 0.08 U/μl of Proteinase K and 1.0 U/μl of RNAse Inhibitor (A&A Biotechnology) with shaking at 37 °C for 5 min and followed by the RNA isolation with TriReagent (Sigma-Aldrich) and chloroform/phenol extraction. Isolated total RNA concentration was measured with the Qubit 4 fluorometer (Invitrogen) and reversely transcribed with the High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific). Gene expression was analyzed from 7.5 ng of cDNA per sample with real-time PCR using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) on QuantStudio 7 Flex (Applied Biosystems). The qPCR data were statistically analyzed with GraphPad Prism software. Relative expression was calculated with ddCt and referred to RPS29 gene expression. The variations in gene expression were determined by two-tailed Student’s t-test (n ≥ 2); P-values were considered significant as follows: additive vs control: Pa < 0.05; Pb < 0.01 and Pc < 0.001; timepoint vs timepoint (within the particular additive group): *P < 0.05; **P < 0.01 and ***P < 0.001. Sequences of primers used are listed in Supp. Tab. 1 .
3
Plasma miRNA Extraction Using Spike-In
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100 µl of plasma was mixed with 1 ml of RNA Extracol (EURx, Poland) and 5 µl of spike-in miRNA standard (5 nM solution of cel-miR-39-3p, MIMAT0000010; Genesius, Poland) until homogeneity. Next, 200 µl of chloroform (POCH, Poland) was added and mixed. After 5 min incubation, the samples were centrifuged at 4°C and 500 µl of the aqueous phase was mixed with 5 µl of GlycoBlue Coprecipitant (15 µg/ml; ThermoFisher Scientific, USA). RNA was precipitated with 505 µl of isopropanol (POCH, Poland) and incubated at -20°C for 20 min, followed by centrifugation at 4°C. The pellet was washed twice with ice cold 75% EtOH (Merk, Germany), air dried and dissolved in 10 µl of Milli-Q water supplied with 1 U/µl RNase inhibitor (A&A Biotechnology, Poland). The benchtop isolation stages were performed on ice.
Michal Prendecki, Jolanta Florczak-Wyspianska, Marta Kowalska, Jan Ilkowski, Teresa Grzelak, Katarzyna Bialas, Wojciech Kozubski, Jolanta Dorszewska
Michal Prendecki, Jolanta Florczak-Wyspianska, Marta Kowalska, Jan Ilkowski, Teresa Grzelak, Katarzyna Bialas, Wojciech Kozubski, Jolanta Dorszewska
4
Plasma miRNA Quantification Protocol
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We applied 2 µl of isolated RNA for simultaneous single tube polyadenylation/reverse transcription reaction (PA-RT, 0.3 U Poly-A polymerase, EURx; 40 U of TranScriba MMLV reverse transcriptase, 10 U of RNase inhibitor; A&A Biotechnology, Poland) with the mixture of 0.5× Poly-A and 0.5× MMLV reaction buffers, supplemented with MnCl 2 , dNTPs and 3'RACE adaptor as primer (Genesius, Poland; final concentrations: 1.9 mM, 1 mM and 0.5 µM, respectively) in volume of 10 µl, on ice. The obtained cDNA was diluted to 200 µl with Milli-Q water and 2 µl was used for qPCR assessment of miRNAs relative content using complementary DNA primers (final concentration 0.25 nM) and 1× SsoFast EvaGreen Supermix (BIO-RAD, USA) in final volume of 10 µl on CFX-Connect Real-Time System (BIO-RAD, USA), as previously reported [3] . Oligos' sequences and cycling conditions are shown in Tables I andII. The Ct values and relative abundance of circulating plasma miRNAs were calculated with respect to spike-in RNA standard by ΔCt method, as described before [54] .
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