The RNA extraction kit is a laboratory tool designed to isolate and purify RNA from a variety of biological samples, such as cells, tissues, or body fluids. The kit employs a chemical-based method to lyse the samples and selectively capture the RNA molecules, which are then washed and eluted for further analysis or experimentation.
The REV strain SNV (GenBank: DQ003591.1) was kindly provided by Professor Shuhong Sun and Zhizhong Cui from Shandong Agricultural University. Spleen tissues were collected in our previous SPF chickens experiment (Gao et al. 2019a) . Total RNA was extracted using RNA extraction kit (CWBIO, Beijing, China).
Jiang H., Gao S., Mao M., Điao Y., Tang Y, & Hu J. (2021). The expression profile of miR-222b-5p/MAPK10 in spleens of SPF chickens infected with REV-SNV at 28-42 dpi. Polish journal of veterinary sciences, 24(3).
We extracted total RNA from cells by an RNA extraction kit (CWBIO, Beijing, China). The reverse transcription was then conducted using a HiFiScript cDNA Synthesis Kit (CWBIO). KIF21B mRNA expression was measured by a SYBR Premix Ex Taq II kit (Takara, Japan). The obtained data was analyzed using 2-ΔΔCt method. β-actin was used as the control. The primers used in this study were synthesized by GENEWIZ (Suzhou, China) and as follows: KIF21B forward, 5'-CGA GGA GAC GGA TGA GAA CG-3'; reverse, 5'-CCA CCA GGC TCT CTT CAC TG-3'; β-actin forward, 5'-CCC GAG CCG TGT TTC CT-3', reverse, 5'-GTC CCA GTT GGT GAC GAT GC-3'.
Sun Z., Pan F., Shao J., Yan Q., Lu L., & Zhang N. (2020). Kinesin superfamily protein 21B acts as an oncogene in non-small cell lung cancer.
From ccRCC tissues or cells, total RNA was extracted using an RNA extraction kit (Cwbio, Beijing, China). Afterwards, RNA was purified using phenol–chloroform extraction method. After reversely transcribed using Evo m-mlv reverse transcription reagent (Accyrate Biology, Changsha, China), qRT-PCR was conducted employing SYBR green kit (Accyrate Biology) under the Real-time PCR System (APPlied Biosystems, Foster City, CA, USA). The used primers were shown in Table 1. GAPDH (circ_0101692 and FN1) and U6 (miR-384) were used for normalization and the 2−ΔΔCt technique was utilized to analyze the target gene expression.
Real-time PCR primer synthesis list.
Table 1
Gene
Sequences
circ_0101692
Forward
5, -CAGAAATTCCTGCAGACATCC-3
Reverse
5′-TGATGCAGCGACCATCAC-3′
miR-384
Forward
5, -TGTTAAATCAGGAATTTTAA-3
Reverse
5′-TGTTACAGGCATTATGAA-3′
FN1
Forward
5, -ACCCTGGGTATGACACTGGA-3,
Reverse
5, -TGCCTCTGCTGGTCTTTCAG-3,
U6
Forward
5, -CTCGCTTCGGCAGCACA-3,
Reverse
5, -AACGCTTCACGAATTTGCGT-3,
GAPDH
Forward
5, -AGAAAAACCTGCCAAATATGATGAC-3,
Reverse
5, -TGGGTGTCGCTGTTGAAGTC-3,
Zhang H, & Ma M. (2023). Circ_0101692 knockdown retards the development of clear cell renal cell carcinoma through miR-384/FN1 pathway. Translational Oncology, 28, 101612.
An RNA extraction kit (Cwbio, China) was used to obtain the total RNA. SYBR green Kit from Acyrate Biology was used to do qRT-PCR after reverse transcription by mean of Evo m-mlv reverse transcription reagent acquired from Accyrate Biology, China. 95 °C for 5 min, 40 cycles of 95 °C for 15 s, 56 °C for 30 s, and 72 °C for 20 s made up the reaction conditions. Table 1 lists the primers that were utilized. The expression levels were normalized with GAPDH and U6. The levels of mRNA and miRNA expression were determined using the 2−ΔΔCt technique.
Real-time PCR Primer synthesis list.
Table 1
Gene
Sequences
RBM15
Forward
5，-CTTGGCGCTGACCCTGTTAT-3，
Reverse
5，-TCATCCGCTGTTCAACCAGT-3，
KRT4
Forward
5，-GGCTCCTTCAGTGGTAAGGG-3，
Reverse
5，-GGTGAGCAAGCTCTGGTTGA-3，
U6
Forward
5，-CTCGCTTCGGCAGCACA-3，
Reverse
5，-AACGCTTCACGAATTTGCGT-3，
GAPDH
Forward
5，-AGAAAAACCTGCCAAATATGATGAC-3，
Reverse
5，-TGGGTGTCGCTGTTGAAGTC-3，
, & Wang H. (2024). The RNA m6A writer RBM15 contributes to the progression of esophageal squamous cell carcinoma by regulating miR-3605-5p/KRT4 pathway. Heliyon, 10(2), e24459.
Total RNA was extracted from primary cardiofibroblasts by using RNA Extraction Kit (CWBIO, Beijing, China). Applied Biosystems cDNA Reverse Transcription and CWBIO SYBR RT-PCR kits were used for quantitative real-time RT-PCR. The primer sequences for ADAM17, ACE2 and β-actin genes are listed in Supplementary Table S1. Quantitative values were obtained from the threshold cycle value (Ct value) and relative mRNA expression levels were analyzed by the 2-△△Ct method.
Cheng J., Xue F., Cheng C., Sui W., Zhang M., Qiao L., Ma J., Ji X., Chen W., Yu X., Xi B., Xu F., Su G., Zhao Y., Hao P., Zhang Y, & Zhang C. (2022). ADAM17 knockdown mitigates while ADAM17 overexpression aggravates cardiac fibrosis and dysfunction via regulating ACE2 shedding and myofibroblast transformation. Frontiers in Pharmacology, 13, 997916.
Total RNA were extracted with an RNA extraction Kit (CWBio, Beijing, China) and quantified. Equivalent amounts of RNA (1 μg) were used for reverse transcription (Vazyme, Nanjing, China). qPCR was performed using SYBR qPCR Mix (Vazyme) and gene specific primers (IL-6, IL-1β, IL-8, TLR2, TLR4 and GAPDH, Table 1) according to manufacturer’s instruction. Expression of target genes were normalized to GAPDH.
Sun M., Ji Y., Li Z., Chen R., Zhou S., Liu C, & Du M. (2020). Ginsenoside Rb3 Inhibits Pro-Inflammatory Cytokines via MAPK/AKT/NF-κB Pathways and Attenuates Rat Alveolar Bone Resorption in Response to Porphyromonas gingivalis LPS. Molecules, 25(20), 4815.
We used the GenBank SUMO1 (NM009460.2) and Ubc9 (NM_001177610) gene sequences to design specific RNAi fragments (Table 1). The polymerase chain reaction (PCR) primers were designed using Premier 5.0 software (Table 2). Real-time PCR (RT-PCR) primers for mouse SUMO1 and Ubc9 genes were designed according to their GenBank sequences (Table 3). Total RNA was isolated from RAW264.7 cells using an RNA extraction kit (CW Biotech, China) and was reverse transcribed into complementary DNA (cDNA) using the HiFi-Script first-chain synthesis kit (CW Biotech). SUMO1 and Ubc9 genes were PCR-amplified using cDNA as the template. Each target gene was individually ligated into pMD19-T and, after transformation into Escherichia coli, positive colonies were picked. The recombinant plasmids containing the SUMO1 or Ubc9 genes were purified. The gene fragments were then isolated by HpaI and XhoI digestion and then individually ligated into the pLEX-MCS overexpression vector to generate pLEX-SUMO-1 and pLEX-Ubc9. We also constructed 2 RNAi vectors, pLL3.7-SUMO-1 and pLL3.7-Ubc9, using a similar subcloning strategy.
Yi J., Wang Y., Li Q., Zhang H., Shao Z., Deng X., He J., Xiao C., Wang Z., Wang Y, & Chen C. (2019). Interaction between Brucella melitensis 16M and small ubiquitin-related modifier 1 and E2 conjugating enzyme 9 in mouse RAW264.7 macrophages. Journal of Veterinary Science, 20(5), e54.
HEK-293FT cells and RAW264.7 cells were transfected with the lentiviral knockdown or overexpression vectors using Lipofectamine 2000 (Invitrogen, USA) (5 mL of plasmid was added to 2 × 105 cells). The plasmids and cells were mixed thoroughly and placed in a 37°C 5% CO2 incubator. K-S, O-S, SUMO1 overexpression-knockdown (OS-KS), K-U, O-U, and Ubc9 overexpression-knockdown (OU-KU) groups were generated. After incubation for 48 h, total RNA from each group was extracted using an RNA extraction kit (CW Biotech). The cDNA was synthesized from the mRNA by using AMV reverse transcriptase (CW Biotech), following the manufacturer's instructions. The stable cell lines were verified by quantitative RT-PCR (qRT-PCR).
Yi J., Wang Y., Li Q., Zhang H., Shao Z., Deng X., He J., Xiao C., Wang Z., Wang Y, & Chen C. (2019). Interaction between Brucella melitensis 16M and small ubiquitin-related modifier 1 and E2 conjugating enzyme 9 in mouse RAW264.7 macrophages. Journal of Veterinary Science, 20(5), e54.
Total RNA from cultured cells was extracted with RNA extraction kit (Aidlab). For RNA extraction from tissues, 20 mg tissues were used and total RNA was extracted with RNA extraction kit (CWBIO). Approximately 1 μg of total RNA was used for reverse transcription with a first strand cDNA synthesis kit (Toyobo or Vazyme). The resulted cDNA was then assayed with quantitative PCR. β-actin was used for normalization. The sequences of primers are in Supplementary Table 11. Assays were repeated at least three times. Data were shown as average values ± SD or SEM. P value was calculated using student’s t test.
Xiao Q., Wang C.Y., Gao C., Chen J.D., Chen J.J., Wang Z., Ju L.G., Tang S.B., Yao J., Li F., Li L.Y, & Wu M. (2022). Regulation of KDM5C stability and enhancer reprogramming in breast cancer. Cell Death & Disease, 13(10), 843.
Total RNA was isolated from CC tissues or cells using an RNA extraction kit (CWBio, Beijing, China). cDNA was created using Evo m-mlv reverse transcription reagent (Accyrate Biology, Changsha, China). qRT-PCR was performed using the SYBR Green kit (Accyrate Biology) and a Real-time PCR System (Applied Biosystems, Foster City, CA, USA). RNase R was used to degrade circ_0101050 in linear UGGT2. The extracted RNA was treated with RNase R (3 U/μg, Epicentre Technologies, Chicago, IL, USA) for 1 h at 37 °C. For the subcellular location assay, cytoplasmic and nuclear RNAs from SW480 and SW620 cells was isolated using a PARIS Kit (Thermo Fisher Scientific, Waltham, MA, USA). The 2−ΔΔCt method was used to calculate the gene expression, and GAPDH and U6 were used as standards. The primers used are listed in Table 1.
Real-time PCR Primer synthesis list.
Table 1
Gene
Sequences
circ_0101050
Forward
5, -GTGGTGTGAAACCTGGTGTG-3,
Reverse
5, -TTGAGTATCTTGCTTTTGAAGCTG-3,
miR-140–3p
Forward
5, -TACCACAGGGTAGAACCACGG-3,
Reverse
5, -GCAGGGTCCGAGGTATTC-3,
MELK
Forward
5, -AAAGGTCAAACTTGCCTGCC-3,
Reverse
5, -AGTACTTGGTAAACTTGATGTCTGT-3,
U6
Forward
5, -CTCGCTTCGGCAGCACA-3,
Reverse
5, -AACGCTTCACGAATTTGCGT-3,
GAPDH
Forward
5, -AGAAAAACCTGCCAAATATGATGAC-3,
Reverse
5, -TGGGTGTCGCTGTTGAAGTC-3,
Cheng K., Chen H., Chen B., Li J., Fan C., Yan H., Huang W., Zhao T., Luo Y, & Peng L. (2024). Hsa_circ_0101050 accelerates the progression of Colon cancer by targeting the miR-140–3 p/MELK axis. Translational Oncology, 44, 101890.
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