cDNA was synthesized using 0.5µg of tissues total RNA and the MMLV reverse transcription Kit, following the manufacturer’s protocol (Promega, Charbonière, France). Gene expression was quantified using real-time PCR with an Applied Biosystems 7300 real-time PCR system. RT-qPCR primers were synthesized, as shown in Table S1 . Real-time PCR was performed using 2 μL of cDNA with specific primers targeting the genes of interest and 400 nM of each primer and 4 µL of Fast Eva Green Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.) in an 8 µL reaction volume. The cycling conditions were 45 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 20 s. mRNA expression (fold induction) was quantified by calculating the 2−ΔΔCT value, with actin mRNA as an endogenous control and the mosquitoes challenged with uninfectious blood meal as control.
M mlv reverse transcription kit
Manufactured by Promega
Sourced in United States, France
The M-MLV Reverse Transcription Kit is a laboratory product designed for the reverse transcription of RNA into complementary DNA (cDNA). It contains the M-MLV Reverse Transcriptase enzyme, necessary buffers, and other essential components required for the reverse transcription process.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (25 mentions)
Trizol, by Thermo Fisher Scientific (21 mentions)
Sybr premix ex taq, by Takara Bio (5 mentions)
Rnase free dnase 1, by Promega (4 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Tri reagent, by Merck Group (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Fast eva green master mix, by Thermo Fisher Scientific (2 mentions)
Abi prism 7500 system, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Genematrix universal rna kit, by EURx (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Cfx maestro software, by Bio-Rad (2 mentions)
Cfx connect real time system, by Bio-Rad (2 mentions)
Sybr green real time pcr master mix, by Promega (2 mentions)
Ex taq, by Takara Bio (2 mentions)
Mx3000p real time pcr system, by Agilent Technologies (2 mentions)
Gotaq green master mix, by Promega (2 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (2 mentions)
Rq1 rnase free dnase, by Promega (2 mentions)
79 protocols using m mlv reverse transcription kit
1
Quantification of Gene Expression in Mosquitoes
2
Validating miR-24 and YKL-40 Expression
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Validation of the expression of the sequence-specific miR-24 was determined using quantitative stem-loop qRT-PCR. Validation of its target gene YKL-40 was performed using qRT-PCR. U6 and 18S rRNA were used as internal controls for normalization. Briefly, small RNAs were transcribed into cDNA using the designed miRNA specific stem loop-RT primers and the NCode™ miRNA First-Strand cDNA Synthesis Kit (Invitrogen). Then, 500 ng of total RNA was reverse transcribed into cDNA using the M-MLV Reverse Transcription Kit (Promega, USA). All qPCRs (20 μl total reaction) were performed with 5 μL of generated cDNA using the SYBR Green Master Mix protocol (TaKaRa Biotechnology Co., Ltd) with the ABI PRISM 7500 system (Applied Biosystems, Forster City, CA, USA). The reactions were incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 sec, 72°C for 30 sec. The dissociation curve analysis of the PCR products was determined in the final stage of 55°C to 95°C. All reactions were run in triplicate, and each sample was replicated three times. All fold changes using qRT-PCR were determined using the 2−ΔΔCT mean ± SEM [32 (link), 33 ]. All primers are listed in Supplementary Table 2 .
3
Validating RNA-seq Unigene Expression via RT-qPCR
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The expression of identified unigenes by RNA-seq was validated using RT-qPCR. For this, we selected 13 unigenes showing differential expression between control and salt-treated samples. The same samples which were utilized for RNA-seq were used for the RT-qPCR analysis. The total RNA was reverse transcribed into cDNA using M-MLV Reverse Transcription Kit (Promega, USA) according to the manufacturer’s instructions. Gene-specific primers (Supplementary Table S7 ) for RT-qPCR were designed with help of integrated DNA technologies (IDT) software (https://sg.idtdna.com/primerquest/home/index ). A 10 µl reaction was carried out on the Roche real-time PCR system (Roche, Switzerland) with the absolute SYBR Green qPCR Kit Master Mix according to the manufacturer’s instructions. The cycle threshold value (CT) was determined and differential expression was calculated using the 2−ΔΔCT method73 (link) with the TEF gene (PiTEF) of P. indica used as an endogenous reference. Each experiment was performed in triplicate.
4
Cloning and Characterization of Chrysanthemum MYB Transcription Factors
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Total RNA was extracted from the ray florets of chrysanthemum seedlings using the Quick RNA Isolation Kit (Huayueyang Biotechnology Co. Ltd., Beijing, China), and first-strand cDNA was synthesized by the M-MLV Reverse Transcription Kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. All of the CmMYB sequences were obtained from the chrysanthemum RNA-Seq database, which was constructed using the ray florets of the capitulum during different developmental stages and light conditions in our previous study [26 (link)]. The open reading frames of CmMYB4, CmMYB5, CmMYB6, and CmMYB7 were acquired by PCR amplification from the cDNA with specific primers (Table 1 ). Amplification conditions were previously described [26 (link)].
5
Validating Microarray Analysis via qRT-PCR
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To verify the results obtained from the microarray analysis, quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to detect the expression levels of differentially expressed miRNAs. Total RNA from cells was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and then was subjected to reverse transcription reaction to synthesize cDNA using the MMLV reverse transcription kit (Promega, Madison, WI, USA). qRT-PCR was performed with FS Universal SYBR Green Master (Roche, Foster City, CA, USA) in the ABI PRISM 7500 System (Applied Biosystems, Foster City, CA, USA) according to the manufacture's protocol. The bulge-loop miRNA qRT-PCR primer sets (one reverse transcription primer and a pair of quantitative PCR primers for each set) were designed and synthesized by RiboBio (Guangzhou, China). U6 small nuclear RNA was used as an endogenous control. The fold change of miRNA in log 2 scale was calculated by the equation 2−ΔΔCt, where ΔCt = CtmiRNA– CtU6 and ΔΔCt = ΔCttreated samples−ΔCtuntreated controls. Comparisons between groups were done by one-way analysis of variance with Dunnett's post hoc corrections for multiple comparisons. Statistical analysis was performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered significant differences.
6
Morphine Modulates GDNF Expression in IECs
7
RNA Extraction and qRT-PCR for Gene Expression Analysis
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Total RNA from cells and tissues was extracted using TRIzol (Invitrogen, USA) according to the manufacturer’s directions. To quantify the mRNA levels, DNA-free RNA was reverse-transcribed using the M-MLV Reverse Transcription Kit (Promega). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out using Power SYBR® Green PCR Master Mix (Thermo Fisher). All reactions were repeated in triplicate wells. In this study, either GAPDH or human small nuclear RNA U6 was used for normalization. The 2−ΔΔCt method was used for calculation. All the primer pairs are shown in Table 2 .
Primers used in the qPCR experiments
| Primer name | Sequence |
|---|---|
| hsa-miR-362-3p-F | 5’-GCGCGAACACACCTATTCAAG-3’ |
| hsa-miR-362-3p-R | 5’-AGTGCAGGGTCCGAGGTATT-3’ |
| U6 small nuclear 1-F | 5’-CTCGCTTCGGCAGCACA-3’ |
| U6 small nuclear 1-R | 5’-AACGCTTCACGAATTTGCGT-3’ |
| SERBP1-F | 5’-TAGACCGATTATTGACCGACC-3’ |
| SERBP1-R | 5’-TTGACAGTTCCCCAGTTGTG-3’ |
| GAPDH-F | 5’-AATCCCATCACCATCTTC-3’ |
| GAPDH-R | 5’-AGGCTGTTGTCATACTTC-3’ |
8
Quantitative PCR Analysis of Gene and miRNA Expression
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Total RNA was isolated with GeneMATRIX Universal RNA/miRNA kit (Eurx, Poland), followed by reverse transcription (RT) using M-MLV reverse transcription kit (Promega, Madison, WI, USA). Blank qPCR Master Mix kit (EURx, Poland) with specific TaqMan probes (Thermo Fisher Scientific, USA) were used (Table 1 ). Reverse transcription of miRNA was performed using the NCode VILO miRNA cDNA Synthesis Kit (Invitrogen), according to the manufacturer’s protocol. For the evaluation of miRNA expression by quantitative real-time PCR, SYBR Green qPCR Master Mix (EURx) and universal reverse primer from the NCode VILO miRNA cDNA Synthesis Kit (Invitrogen) were used with the indicated forward primers designed according to the kit’s instruction (primers are listed in Supplementary materials). The ΔCt method (2-∆Ct) was used to calculate relative expression of the genes, using GAPDH as a relative control for mRNA and U6 snRNA as a control for miRNA.
Taq-Man qRT-PCR probes list.
| Gene name | Cat no. | Gene name | Cat no. |
|---|---|---|---|
| PRKAA1 | Hs01562315_m1 | VIM | Hs00958111_m1 |
| PRKAA2 | Hs00178903_m1 | GFP | Mr03989638_mr |
| PRKAB1 | Hs00272166_m1 | DSP | Hs00950591_m1 |
| PRKAG1 | Hs01091629_g1 | CLDN1 | Hs00221623_m1 |
| SNAI1 | Hs00195591_m1 | OCLN | Hs00170162_m1 |
| SNAI2 | Hs00161904_m1 | DES | Hs00157258_m1 |
| CDH1 | Hs01023894_m1 | MMP-2 | Hs00234422_m1 |
| ZEB-1 | Hs00232783_m1 | ACTB | Hs99999903_m1 |
| MMP-1 | Hs00899658_m1 | MMP-13 | Hs00233992_m1 |
| mGAPDH | Mm99999915_g1 | hGAPDH | Hs02786624_g1 |
9
qPCR Analysis of Inflammatory Markers
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Total RNA was isolated using the Trizol reagent (Sigma) method. The yield of RNA was subjected to reverse transcription using the MMLV reverse transcription kit (Promega). Then, cDNA was subjected to qPCR for NLRP3, caspase-1, IL-1β, IL-18 and the β-actin gene using the SYBR Green PCR kit (Promega). Quantification was done by normalization against β-actin. The following primer sequences were used: β-actin upstream primer: 5’-CGTGACATTAAGGAGAAGCTG-3’, downstream primer: 5’-CTAGAAGCATTTGCGGTGGAC-3’; NLRP3 up-stream primer: 5’-CTTCCTTTCCAGTTTGCTGC-3’, downstream primer: 5’-TCTCGCAGTCCACTTCCTTT-3’; caspase-1 upstream primer: 5’-GCCCAAGTTTGAAGGACAAA-3’, downstream primer: 5’-GGTGTGGAAGAGCAGAAAGC-3’; IL-1β upstream primer: 5’-GCCCTAAACAGATGAAGTGCTC-3’, downstream primer: 5’-GAACCAGCATCTTCCTCAG-3’; IL-18 upstream primer: 5’-GCTTGAATCTAAATTATCAGTC-3’, downstream primer: 5’-GAAGATTCAAATTGCATCTTAT-3’.
10
Quantitative mRNA Expression Analysis
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Gene expression on the level of mRNA was analyzed by RT-PCR. Briefly, total RNA was isolated with GeneMATRIX Universal RNA kit (Eurx, Gdańsk, Poland). Reverse transcription (RT) was performed with MMLV reverse transcription kit (Promega, Madison, WI, USA) according to manufacturer instructions. PCR reactions were performed with Taq Master Mix kit (Eurx, Gdańsk, Poland); 100 ng of generated cDNA was used per reaction. All primers used were designed to work optimally at an annealing temperature of 55 °C. Primer sequences have been collected in the Table 1 .
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