RNA extraction and quantitative PCR gene expression were measured using quantitative reverse transcription polymerase chain reaction. Each treatment was replicated with four biological repeats and four technical repeats. The RNeasy Mini Kit (Qiagen, Dusseldorf, Germany) was used to isolate total RNA from the leaves (0.05 g from samples stored at -70°C), and 1 μg of RNA was used to generate cDNA. We used real-time quantitative PCR (qPCR) to determine the mRNA levels according to a modified method that was described previously (Guo et al., 2018 (link)). Specific primers for each gene were designed from the expressed sequence tag sequences using Primer 5 software (Table S.7
Sybr premix ex taq
Manufactured by Qiagen
Sourced in United States, Germany
SYBR Premix Ex Taq is a ready-to-use master mix for real-time PCR amplification. It contains SYBR Green I dye, Taq DNA polymerase, dNTPs, and buffer components.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (15 mentions)
High capacity cdna reverse transcription kit, by Qiagen (4 mentions)
Lightcycler 480, by Roche (2 mentions)
Exorneasy midi kit, by Qiagen (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Primer express, by Thermo Fisher Scientific (1 mentions)
Miscript reverse transcription kit, by Qiagen (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rnase r, by Illumina (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Miscript hispec buffer, by Qiagen (1 mentions)
Miscript 2 reverse transcription kit, by Qiagen (1 mentions)
Miscript sybr green real time pcr kit, by Qiagen (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
Transscript green one step qrt pcr supermix, by Qiagen (1 mentions)
Taqman reverse transcription reagents kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Revertra ace, by Toyobo (1 mentions)
21 protocols using sybr premix ex taq
1
Quantitative PCR Gene Expression Analysis
2
Validating Transcriptome Regulatory Relationships
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To validate the transcriptome regulatory relationship between the DEGs and DELs, cDNA from the temperature treatment and fasting/re-feeding treatment samples were all used for qRT-PCR. The primer sets were designed using Primer Express software (Applied Biosystems, USA), and the sequences were shown in Additional file 1 : Table S10 . All real-time PCRs were carried out in a LightCycler 480 real-time PCR machine (Roche, Switzerland) with a mixture of 5 µL 2×SYBR Premix ExTaq (Qiagen, Germany), 1.0 µL of diluted cDNA, 3 µL of PCR-grade water, and 0.5 µL of each 10 µM primer. The PCR was initiated by denaturation at 95 °C for 30 s; followed by 40 amplification cycles at 95 °C for 15 s and 60 °C for 30 s. Dissociation protocols were used to measure the melting curves. The relative expression level was calculated with the 2−ΔΔCt method [80 (link)], and the data were expressed as the mean ± SD. Statistical significance was determined by the one-way ANOVA and Student’s t-test for multiple groups and two groups comparison, and P < 0.05 was considered statistically significant.
3
EphA2 Expression Quantification by RT-qPCR
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TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for total RNA extraction from all types of cells according to standard procedures. The detection of EphA2 was performed via RT-qPCR using a high-capacity cDNA Reverse Transcription kit (Qiagen AB) and SYBR Premix Ex Taq (Qiagen AB). The conditions for cDNA synthesis were as follows: 42°C for 30 min and 85°C for 5 sec. The thermocycling conditions were as follows: 95°C for 3 min; 39 cycles of 95°C for 5 sec, 56°C for 10 sec, 72°C for 25 sec; 65°C for 5 sec; 95°C for 50 sec. The relative expression levels were normalized to endogenous control GAPDH and were expressed as 2−ΔΔCq (22 (link)). The sequences of the primers were as follows: EphA2 forward, 5′-CTGGTCTGCAAGGTGTCTGA-3′ and reverse, 5′-TTGGACAACTCCCAGTAGGG-3′; and GADPH forward, 5-GATATTGTTGCCATCAATGAC-3 and reverse 5-TTGATTTTGGAGGGATCTCG-3.
4
Isolation and Expression Analysis of RNAs
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Trizol reagent (Invitrogen) was utilized to isolate total RNAs from RB tissue and cells according to the standard introductions. For mRNA expression detection, the PrimeScript™ RT Reagent Kit (Takara, Dalian, China) was utilized to synthesize the complementary DNA (cDNA). For miRNA analysis, cDNA was generated using the miScript Reverse Transcription kit (Qiagen, Valencia, CA, USA). Then qPCR was performed with SYBR Premix Ex Taq (Qiagen). The fold change of miRNA or mRNA was calculated using 2−ΔΔCT methods and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6. All experiments were performed in triplicate. The special primers for miR-183-5p or U6 were purchased from GeneCopoeia. The primer sequences for SNHG16, NRAS and GAPDH are listed as followed: SNHG16 F, 5’-CAGAATGCCATGGTTTCCCC-3’, and R 5’-TGGCAAGAGACTTCCTGAGG-3’; NRAS F, 5’-ATGAGGACAGGCGAAGGCT-3’, and R, 5’-TGAGTCCCATCATCACTGCTG-3’;GAPDH F, 5’-ATTCCATGGCACCGTCAAGGCTGA-3’, and R, 5’-TTCTCCATGGTGGTGAAGACGCCA-3’.
5
Exosomal RNA Extraction and Analysis
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Whole-RNA extracts were prepared using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and total RNA from exosomes was isolated with the exoRNeasy Midi Kit (Qiagen, Valencia, CA, USA) according to the standard procedure. Then extracted RNA was interacted with Rnase R (Epicentre, Madison, WI, USA), followed by incubation with RNeasy MinElute Cleanup Kit (Qiagen). RNA samples were reversely transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Qiagen), and cDNA amplification was carried out with SYBR Premix Ex Taq (Qiagen). Glyceraldehyde 3-phosphate dehydrogenase (GADPH) and U6 small nuclear B noncoding RNA (U6) were used as internal references to normalize the fold changes using 2−ΔΔCt method. The specific primer sequences were listed as follows:
6
Mouse Endometrial RNA Extraction and qPCR
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Total RNA was extracted from mouse endometrial tissues or cultured cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. RT of cDNA was conducted using the miScript II Reverse Transcription kit with miScript HiSpec Buffer (Qiagen, Inc., Valencia, CA, USA,). Briefly, RNA was mixed with miScript HiSpec Buffer, RNAse-free water and miscript Reverse Transcriptase Mix, and was incubated at 37°C for 60 min and 95°C for 5 min. qPCR was conducted with the miScript SYBR-Green Real Time PCR kit (Qiagen, Inc.) for miRNA detection and SYBR Premix Ex Taq. The specific primers for mmu-miR-96, U6, Bcl2, decidual/trophoblast prolactin-related protein (dtPRP) and β-actin were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) (Table I ). The qPCR master mix (15 µl) contained 7.5 µl SYBR Premix Ex Taq, 0.6 µl primers, 1.2 µl cDNA and 5.1 µl diethylpyrocarbonate-treated H2O. The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec; 40 cycles at 95°C for 5 sec (denaturation) and 60°C for 30 sec, followed by 72°C for 5 sec. Experiments were performed in triplicate. Data obtained from qPCR were analyzed using the 2-ΔΔCq method (26 (link)).
7
Quantification of NNT-AS1, FOXM1 and miR-22
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Total RNA was extracted using a TRIzol kit (Invitrogen) according to the standard protocol. For NNT-AS1 and FOXM1 detection, total RNA was reversely transcribed into complementary DNA (cDNA) using a high-capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA), and then qPCR was carried out with SYBR Premix Ex Taq (Qiagen, Valencia, CA, USA). As to miR-22 detection, cDNA was synthesized using a TaqMan Reverse Transcription Reagents Kit (Applied Biosystems) and amplified with TransScript Green One-Step qRT-PCR SuperMix (Qiagen). The relative expression was analyzed by the 2-△△Ct method and normalized byglyceraldehyde-3-phosphate dehydrogenase (GADPH) or U6 small nuclear RNA (snRNA). The specific primer sequences used were as follows: NNT-AS1, F: 5′-TCTCCTAAGTCGAGGACTAGC-3′, R: 5′-AGGCACTCACTAGCATCACGCT-3′;FOXM1, F: 5′-GGAGCAGCGACAGGTTAAGG-3′, R: 5′-GTTGATGGCGAATTG TATCATGG-3′; miR-22, F: 5′-GGGGGATCCCTGGGGCAGGACCCT-3′, R: 5′-GGGGAATTCAACGTATCATCCACCC-3′; U6: F:5′-GCTTCGGCAGCACATATACTAAAAT-3′, R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′; GAPDH, F: 5′-AACTTTGGCATTGTGGAAGG-3′, R: 5′-ACACATTGGGGGTAGGAACA-3′.
8
qRT-PCR Analysis of Skeletal Muscle Gene Expression
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Total RNA was extracted from freshly isolated GA muscles using TRI Reagent (Invitrogen) and analyzed by qRT-PCR using Rotor-Gene-Q pure-detection software. The first-strand complementary DNA was performed synthesized from 1 μg of RNA using Promega’s RT system according to the manufacturer’s instructions (Promega, Madison, WI, USA). qRT-PCR (Qiagen) was performed with SYBR Green technology (SYBR Premix Ex Taq, Qiagen) using specific primers against indicated genes. Expression levels were determined using the 2−ΔΔCt method and normalized to the housekeeping gene GAPDH. Primers are listed in Supplementary Table 3 .
9
Quantification of mRNA Levels
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Total RNA was extracted from the brain, liver, spinal cord L4, tibialis anterior muscle, freshly isolated FAPs, and chondrocytes using a TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) and analyzed by qRT-PCR. First-strand complementary DNA was synthesized from 1 μg of RNA using ReverTra Ace (Toyobo, Osaka, Japan) containing random oligomer according to the manufacturer’s instructions. qRT-PCR (QIAGEN) was performed with SYBR Green technology (SYBR Premix Ex Taq, QIAGEN) using specific primers against indicated genes. Relative mRNA levels were determined using the 2−ΔΔCt method and normalized to Gapdh (Figure 1H , Figure 1—figure supplement 1K ). Primers are listed in Supplementary file 1 .
10
Exosome RNA Extraction and qRT-PCR Analysis
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The extraction of whole-RNA from exosome pellets, cells, or tissues was performed by TRIzol reagent (Invitrogen) according to the standard procedure. Then Prime Script RT Master Mix and SYBR Premix Ex Taq (Qiagen, Valencia, CA, USA) were utilized for cDNA generation and qRT-PCR analysis, respectively. The relative expression was assessed using 2−ΔΔCt method with the internal references of GADPH and U6. The sequence of primers was listed: circ_0008717: F 5’-GCCTTTCCATTCCGTCAGGA-3’, R 5’-ACCACGCTCAAAACAAAGGTG-3’; miR-1287-5p: F 5’-AGCTGGATCAGTGGTTCGAG-3’, R 5’-CAGTGCAGGGTCCGAGGTAT-3’; PAK2: F 5’-TCTTCCTCCCCCAGGGTTG-3’, R 5’-AATCGAGCCCACTGTTCTGG-3’; GAPDH: F 5’-GAAGGTGAAGGTCGGAGTC-3’, R 5’-GAAGATGGTGATGGGATTTC-3’; U6: F 5’-GCTTCGGCAGCACATATACTAAAAT-3’, and R 5’- CGCTTCACGAATTTGCGTGTCAT-3’.
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