As the expression of cardiac genes in the case and control groups was intended to be quantified in 2 days (days 3 and 7), MSCs were cultured in two pairs of T25 culture flasks. MSCs were first exposed to 5-azacytidine for 24 h. After this period of cardiogenic induction, cells were treated with either 100 nM nicotine or the same volume of PBS as the case and control groups. On days 3 and 7, the appearance of cells in both the case and control groups was monitored by inverted microscopy. On the corresponding days, the culture medium was removed, and cells were washed with PBS and treated with trypsin/EDTA. Following centrifugation of the suspension, the cell pellet was yielded and subjected to RNA extraction (Rnx Plus Solution, CinnaGen Co). The quality and quantity of RNA were examined by NanoDrop. Using the reverse transcriptase enzyme, cDNA was synthesized (AddScript cDNA Synthesis Kit, addbio), and then qPCR (RealQ Plus 2x Master Mix Green, Ampliqon) was performed using cardiac-specific primers of GATA4 and troponin (Table 1 ). This experiment was repeated two times. Differential gene expression was revealed between the case and control groups on days 3 and 7. All the experiments were performed three times.
Rnx plus solution
Manufactured by CinnaGen
Sourced in Iran, Islamic Republic of
RNX-Plus solution is a lab reagent designed for the extraction and purification of RNA from various biological samples. It is a specialized solution that facilitates the separation and isolation of RNA from DNA, proteins, and other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Realq plus 2x master mix green, by Ampliqon (2 mentions)
Revertaidtm first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Expand reverse transcriptase, by Merck Group (1 mentions)
Rotor gene 6000 system, by Qiagen (1 mentions)
Revertaidtm reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Gel doc, by Uvitec (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
First aid reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Abi steponeplus system, by Thermo Fisher Scientific (1 mentions)
Nanodrop, by Eppendorf (1 mentions)
21 protocols using rnx plus solution
1
Nicotine Modulation of Cardiac Gene Expression
2
RNA Extraction and cDNA Synthesis Protocol
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Total RNA was extracted using an RNX-Plus solution (Cinnagen, Iran) kit based on the manufacture’s protocol, with some modifications. Briefly, from each sample, 1 ml was transferred to a sterile 1.5 mL microtube and centrifuged at 6708 × g for 5 minutes. The pellet was washed three times with phosphate-buffered saline and centrifuged at 1677 × g for 5 minutes. Then, 200 µL of ice-cold RNX-Plus lysis buffer were added to each tube, homogenized by vortexing for 10 seconds, and incubated at 70°C for 30 minutes. The vortex process was repeated every 5 minutes (15 (link)). Subsequent RNA extraction steps followed the manufacturer’s instructions. After extraction, each sample was treated with DNase I (Thermo Scientific, U.S.) according to the manufacturer’s instructions. RNA integrity was analyzed by electrophoresis on a 1% agarose gel (Cinnagen, Iran). RNA quantification was performed by measuring the absorbance at 260 nm. Nucleic acid purity was assessed by measuring the ratio of the absorbance at 260 nm and 280 nm. Extracted RNAs were stored at -70°C until required for the experiments. The cDNA synthesis of each sample was measured using a cDNA synthesis kit (Thermo Scientific, U.S.), following the manufacturer’s instructions. All the samples were stored at -20°C until used in the analysis.
3
Testicular Total RNA Extraction
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Total RNA was extracted
from testes using the RNX Plus solution (Cinnagen, Tehran, Iran) according
to the manufacturer’s instructions. The RNX Plus solution (1
mL) was poured onto the testes followed by incubation at room temperature
for 5 min after which 200 μL of chloroform (Merck, Germany)
was added to the mixture. The mixture was shaken vigorously and then
centrifuged at 12 000g for 15 min at 4 °C.
The supernatant was transferred to another microtube to which its
equal volume of isopropanol (Merck, Germany) was added. The microtube
was incubated in ice for 15 min and centrifuged again under the conditions
of the previous centrifugation. Ethanol (75%, 1 mL) was poured on
the obtained precipitate, and, following vortexing, the mixture was
centrifuged at 7500g for 5 min. The resulting pellet
was dissolved in 50 μL of water that was treated with 0.01%
DEPC (Sigma-Aldrich, St. Louis, MO) and stored at −80 °C.
from testes using the RNX Plus solution (Cinnagen, Tehran, Iran) according
to the manufacturer’s instructions. The RNX Plus solution (1
mL) was poured onto the testes followed by incubation at room temperature
for 5 min after which 200 μL of chloroform (Merck, Germany)
was added to the mixture. The mixture was shaken vigorously and then
centrifuged at 12 000g for 15 min at 4 °C.
The supernatant was transferred to another microtube to which its
equal volume of isopropanol (Merck, Germany) was added. The microtube
was incubated in ice for 15 min and centrifuged again under the conditions
of the previous centrifugation. Ethanol (75%, 1 mL) was poured on
the obtained precipitate, and, following vortexing, the mixture was
centrifuged at 7500g for 5 min. The resulting pellet
was dissolved in 50 μL of water that was treated with 0.01%
DEPC (Sigma-Aldrich, St. Louis, MO) and stored at −80 °C.
4
Molecular Analysis of miR-31 in MKN-45 Cells
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Total RNA was extracted from MKN-45-miR-31 or the control groups using RNX-plus solution (Cat No. RN7713C, Cin-naGen Inc., Tehran, Iran) using the manufacturer's instructions (23) . cDNA synthesis was performed using a kit for mRNA synthesis (Cat No. K1641, Fermentas Life Sciences, Germany) and Expand™ Reverse Transcriptase (Cat No. 11785826001, Sigma-Aldrich, USA) for miRNA synthesis. The real-time PCR reactions were carried out using a Rotor-Gene 6000 system (Corbett, Concorde, NSW, Australia) and the data were normalized with the reference genes SNORD47 for miR-31 and β-actin for E2F6 and SMUG1 (24) . In all experiments, we compared the MKN-45-miR-31 (termed "test"), MKN-45-control vector (termed "control") and parental MKN-45 (termed "control").
5
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted by acid guanidinium-phenol-chloroform procedure using the RNX™-plus solution (CinnaGen, Iran) according to the manufacturer’s protocol. The quality and integrity of extracted RNA were assessed by gel electrophoresis (1% agarose) and spectrophotometry respectively. To remove genomic DNA contamination, total RNA was treated with DNase I (Sigma, USA) at 37˚C for 30 minutes. Three microgram of total RNA was reverse transcribed with oligo dT and random hexamers (MWG, Germany) by RevertAidTM Reverse Transcriptase (Fermentas, Canada) in a total volume of 20 μl according to the manufacturer’s protocols.
6
Evaluating TGF-β and VEGF Gene Expression
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We evaluated TGF-β and VEGF gene expression using the RT-PCR technique. On days 7 and 14, tissue samples were isolated and Ribonucleic acid (RNA) was extracted using RNX Plus Solution (Cinnagen). After a quantitative measurement and a qualitative analysis of the RNA, the complementary deoxyribonucleic acid was synthesized using the PreMixkit kit (Bioneer). Sequences of TGF-β forward and reverse primers were 5-AGGAGACGGA ATACAGGGCT-3 ´ and 5-GGATCCACTTCCAACCC AGG-3, respectively. Sequences of VEGF forward and reverse primers were 5-TACCTCCACCATGCCA AGT-3 and 5-TGCATTCACATTTGTTGTGC-3, respectively. The PCR program consisted of 30 cycles for the following program: First, denaturation was performed at 94 °C for 3 minutes, secondary denaturation at 94 °C for 1 minute, annealing at 57 °C for 1 minute, elongation at 72 °C for 1 minute, and final elongation at 72 °C for 10 minutes. Moreover, 1% agarose gel was used to detect bands associated with the PCR products at 150 volts for 25 minutes. The agarose gel was stained with ethidium bromide. Gel Doc (UVITEC) imaging was used to observe and interpret resultant bands
(18 (link),
9 ).
(18 (link),
9 ).
7
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from stolons of G. glabra using RNX plus solution (CinnaGen, Iran) according to the manufacturer’s instructions. To remove genomic DNA contamination, total RNA was treated with RNase-free DNase. The concentration of RNA was estimated via spectrophotometry. Approximately 6 μg of total RNA from each sample was subsequently subjected to first strand cDNA synthesis using random hexamer primers and a M-MuLV Reverse Transcriptase Kit (CinnaGen, Iran) according to the manufacturer’s instructions.
8
Endometrium Total RNA Extraction
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Total RNA was isolated from epithelial cells of endometrium using the RNX plus solution (Cinnagen, Iran) according to the manufacturer’s instructions and as previously described. The purity and integrity of the extracted RNA were assessed by optical density measurements (260/280 nm ratios) and by visual observation of samples electrophoresed on agarose gels. For elimination of genomic DNA, RNA was treated with RNase-free DNase (Qiagen, Germany)
9
RNA Extraction and Plasmid Contamination Check
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RNA was extracted from transfected and untransfected Huh7.5 cells using RNX plus solution (CinnaGen, Iran) according to the manufacturer’s protocols. In this way total RNA was obtained. To remove plasmid DNA, purified RNA was treated with DNase I (Fermentas, Germany) enzyme as described previously (23 (link)); Briefly, 3μg of RNA with 5 units of DNaseI enzyme and 10 X buffers in 10μl total volume prepared then incubated at 37 °C for 30 min. For inactivation of DNase I enzyme, mixture incubated at 65 °C for 10 min. Moreover, the extracted RNA from transfected cells was examined for contamination with transfected recombinant plasmid by PCR method. Briefly, PCR reaction using specific primers for hbha and mtb32C genes was prepared and extracted RNA was used as template in the mixture. Negative results indicated absence of any plasmid contamination in RNA samples.
The extracted RNA was then used for cDNA synthesis and RT-PCR.
The extracted RNA was then used for cDNA synthesis and RT-PCR.
10
Quantitative Analysis of lncRNA Expression
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Total RNA was isolated from the samples using RNX-Plus solution (Cinnagen, Tehran, Iran) according to the instructions of the manufacturer. The extracted RNA is quantitatively and qualitatively evaluated by nano-drop spectrophotometer (Thermo Fisher Scienti c Inc.,Wilmington, USA) and 0.8% agarose gel. cDNA was prepared by First Aid Reverse Transcription Kit (Fermentas).
Real-time PCR was performed by SYBR Green master mix (Amplicon, Denmark) in the LightCycler96 instruments (Roche Life Science Deutschland GmbH, Sandhofer, Germany). Primer sequences were designed and synthesized by Takapou Zist Company.
The primer sequences were as follows: LINC00961 (forward:5'-CTG TTC TGG ATG GGA GCG AA -3'; reverse: 5'-ACA GTC ACC ACG AAC AGC AC-3'), TPT1-AS1 (forward: 5'-CAC TCC CAG ATC TTC ACT TCA GG -3'; reverse: 5'-AAT TGG AGG CCA GTG CTC TG -3'), SAMMSON (forward: 5'-CCT CTA GAT GTG TAA GGG TAG T-3'; reverse: 5'-TTG AGT TGC ATA GTT GAG GAA-3' ) and beta-actin (forward: 5'-ACAGAGCCTCGCCTTTGC - 3'; reverse: 5'-ATCACGCCCTGGTGCCT - 3'). The difference in genes expression in comparison with the reference gene was determined by the 2^-ΔΔCt formula [16] .
Real-time PCR was performed by SYBR Green master mix (Amplicon, Denmark) in the LightCycler96 instruments (Roche Life Science Deutschland GmbH, Sandhofer, Germany). Primer sequences were designed and synthesized by Takapou Zist Company.
The primer sequences were as follows: LINC00961 (forward:5'-CTG TTC TGG ATG GGA GCG AA -3'; reverse: 5'-ACA GTC ACC ACG AAC AGC AC-3'), TPT1-AS1 (forward: 5'-CAC TCC CAG ATC TTC ACT TCA GG -3'; reverse: 5'-AAT TGG AGG CCA GTG CTC TG -3'), SAMMSON (forward: 5'-CCT CTA GAT GTG TAA GGG TAG T-3'; reverse: 5'-TTG AGT TGC ATA GTT GAG GAA-3' ) and beta-actin (forward: 5'-ACAGAGCCTCGCCTTTGC - 3'; reverse: 5'-ATCACGCCCTGGTGCCT - 3'). The difference in genes expression in comparison with the reference gene was determined by the 2^-ΔΔCt formula [16] .
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