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Oligo dt primer

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Oligo-dT primers are short, synthetic DNA sequences composed of a string of deoxythymidine nucleotides. They are designed to anneal to the poly(A) tail of eukaryotic messenger RNA (mRNA) molecules, allowing for the selective isolation and reverse transcription of mRNA into complementary DNA (cDNA).

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Lab products found in correlation

Rneasy mini kit, by Qiagen (7 mentions) Transcriptor first strand cdna synthesis kit, by Roche (7 mentions) Rneasy kit, by Qiagen (5 mentions) M mlv reverse transcriptase, by Promega (5 mentions) 7300 real time pcr system, by Thermo Fisher Scientific (4 mentions) Step 1 real time pcr system, by Thermo Fisher Scientific (3 mentions) Universal master mix, by Thermo Fisher Scientific (3 mentions) Power sybr green pcr master mix, by Bio-Rad (3 mentions) Deoxynucleotide triphosphate, by Thermo Fisher Scientific (3 mentions) Moloney murine leukemia virus m mulv reverse transcriptase, by Thermo Fisher Scientific (3 mentions) Platinum sybr green qpcr supermix udg kit, by Thermo Fisher Scientific (3 mentions) Lightcycler system, by Roche (3 mentions) Lightcycler 480 2, by Roche (3 mentions) Trizol reagent, by Thermo Fisher Scientific (3 mentions) Trizol, by Thermo Fisher Scientific (3 mentions) Sybr green 1 master, by Roche (2 mentions) Rnasin plus, by Promega (2 mentions) Iq sybr green supermix, by Bio-Rad (2 mentions) Cfx96 system, by Bio-Rad (2 mentions) Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)

41 protocols using oligo dt primer

1

RNA Isolation and qPCR Analysis

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Mid-log-phase yeast cells were collected to extract total RNAs using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s instructions. 2 µg of the RNA samples were then reversed transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit using oligo-dT primer (Roche, Indianapolis, IN, USA). The qPCR experiments were carried out using SYBR Green-based method using the Roche LightCycler 480 System.
Lian J., Schultz C., Cao M., HamediRad M, & Zhao H. (2019). Multi-functional genome-wide CRISPR system for high throughput genotype–phenotype mapping. Nature Communications, 10, 5794.
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2

Quantification of mRNA Levels by Real-Time PCR

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Real-time PCR was performed using standard methods as described (61 (link)). The first-strand cDNA was generated by reverse transcription with Oligo (dT) primer (Roche). To quantify the mRNA levels using real-time PCR, aliquots of first-stranded cDNA were amplified with gene-specific primers and Power SYBR Green PCR Master Mix (Bio-Rad) using a Step-1 Real-Time PCR System (Applied Biosystems). The PCR reactions contained 1µg of cDNA (except the cDNA for the IP, for which 5% of the cDNA was used for each gene examined), Universal Master Mix (Applied Biosystems), and 10 µM of forward and reverse primers in a final reaction volume of 20 µL. The mRNA level of different samples was calculated by the data analysis software built in with the 7300 Real-Time PCR System. For RIP-real time PCR, cDNA from IP and input was used and IP samples were normalized to Input samples. The Sequences of primers are provided in supplementary methods.
Li Y., Stockton M.E., Bhuiyan I., Eisinger B.E., Gao Y., Miller J.L., Bhattacharyya A, & Zhao X. (2016). MDM2 Inhibition rescues neurogenic and cognitive deficits in fragile X mice. Science translational medicine, 8(336), 336ra61.
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3

Osteoblast Gene Expression Analysis

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Osteoblasts cells were seeded in 6-well plates at a density of 1 × 106 cells/well. After 6-day culture, cells were treated with the TPF at concentrations of 0, 50, and 100 ng/ml for 48 h. Total RNA from the cells of each well was isolated respectively using NucleoSpin (Macherey–Nagel, Duren, Germany). RNA aliquots were reverse transcribed to complementary DNAs by using an oligo (dT) primer (Roche), deoxynucleotide triphosphate (dNTP), and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Fermentas, Hanover, MD). The complementary DNA products were subjected to PCR amplification with gene-specific primers for mouse Alp, Osteocalcin and Type 1 collagen [23 (link)]. Real-time RT-PCR amplification was performed using a Light Cycler System (Roche) with a Platinum SYBR Green qPCR Super Mix UDG kit (Invitrogen, Carlsbad, CA).
Al Mamun M.A., Hosen M.J., Khatun A., Alam M.M, & Al-Bari M.A. (2017). Tridax procumbens flavonoids: a prospective bioactive compound increased osteoblast differentiation and trabecular bone formation. Biological Research, 50, 28.
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4

Embryonic RNA Extraction and qRT-PCR

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Total RNA from certain stages of embryos were prepared using the RNeasy kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. One microgram of total RNA was used to generate cDNA using a mix of oligo-dT primer (Roche Applied Science, Branford, CT, USA) and the reverse transcriptase (RTase, Roche) according to the manufacturer’s instructions. Real-time PCR was performed using the LightCycle 96 real-time PCR detection system and SYBR Green I Master (Roche). Real-time RT-PCR primers are listed in Table S1. Relative cDNA amounts were calculated using the LC96 program and normalized to the expression of β-actin based on the ΔΔCt method. All reactions were performed as biological triplicates.
Chang H.W., Wang W.D., Chiu C.C., Chen C.H., Wang Y.S., Chen Z.Y., Liu W., Tai M.H., Wen Z.H, & Wu C.Y. (2017). Ftr82 Is Critical for Vascular Patterning during Zebrafish Development. International Journal of Molecular Sciences, 18(1), 156.
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5

Quantitative gene expression analysis

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Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA, USA) and mRNA was reverse transcribed to cDNA using the oligo-dT primer (Roche Applied Science, Branford, CT, USA) based on the manufacturer’s protocols. Quantitative PCR was performed using reagent SYBR Green Mix (Roche), and qPCR primers used in this study are listed in Table S1. The relative expression of respective genes was analyzed and calculated by the ΔΔCt method by considering β-actin as an internal control. All biological experiments were performed in triplicate.
Lo Y.H., Huang Y.S., Chang Y.C., Hung P.Y., Wang W.D., Liu W., Urade R., Wen Z.H, & Wu C.Y. (2022). GTP-Binding Protein 1-Like (GTPBP1l) Regulates Vascular Patterning during Zebrafish Development. Biomedicines, 10(12), 3208.
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6

Real-time qRT-PCR for Gene Expression

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Total RNA was extracted from cells (RNeasy kit, Qiagen). A total of 1 μg of extracted RNA was annealed with oligo dT primer (Roche Life Science), and reverse-transcribed to cDNA using M-MLV reverse transcriptase (Promega) in the presence of RNase Inhibitor (RNasin Plus, Promega). cDNA was mixed with primers and iQ SYBR Green Supermix (Bio-Rad), and mRNA expression levels were measured by real-time qRT-PCR (CFX96 system, Bio-Rad).
Seo J., Kim H., Min K.I., Kim C., Kwon Y., Zheng Z., Kim Y., Park H.S., Ju Y.S., Roh M.R., Chung K.Y, & Kim J. (2022). Weight-bearing activity impairs nuclear membrane and genome integrity via YAP activation in plantar melanoma. Nature Communications, 13, 2214.
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7

Quantitative analysis of LH and LOX genes

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Total RNA was isolated from the tissue samples using the RNeasy kit (Qiagen). After that, mRNA was reverse-transcribed into cDNA using the oligo-dT primer (Roche). Quantitative real-time RT-PCR was then carried out using Applied Biosystems 7500 real-time RT-PCR system (Applied Biosystems, Foster City, CA) using TaqMan gene expression assays (Applied Biosystems) for LH1 (assay ID Hs00609368_m1), LH2 (assay ID Hs00168688_m1), LH3 (assay ID Hs01126612_m1), LOX (assay ID Hs00184700_m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (assay ID Hs99999905_m1). The amount of RT-PCR product for the gene of interest was normalized to the quantity of GAPDH. Each assay was performed in triplicate.
Kamel M., Wagih M., Kilic G.S., Diaz-Arrastia C.R., Baraka M.A, & Salama S.A. (2017). Overhydroxylation of Lysine of Collagen Increases Uterine Fibroids Proliferation: Roles of Lysyl Hydroxylases, Lysyl Oxidases, and Matrix Metalloproteinases. BioMed Research International, 2017, 5316845.
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8

Quantitative RT-PCR Analysis of Gene Expression

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Total RNA was purified using the RNeasy Mini kit (Qiagen). mRNA was reverse transcribed to cDNA using Reverse Transcriptase and oligo-dT primer (Roche) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using a LightCycle 96 instrument (Roche) with SYBR Green I Master (Roche). qRT-PCR primers are listed in Table S1 in File S1. Relative gene expression levels were analyzed by the ΔΔ Ct method, with elongation factor 1α (EF1α) as a reference gene. All reactions were performed as biological triplicates.
Wu B.J., Chiu C.C., Chen C.L., Wang W.D., Wang J.H., Wen Z.H., Liu W., Chang H.W, & Wu C.Y. (2014). Nuclear Receptor Subfamily 2 Group F Member 1a (nr2f1a) Is Required for Vascular Development in Zebrafish. PLoS ONE, 9(8), e105939.
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9

Evaluating RNA Integrity via RT-PCR

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Real-time reverse transcription (RT)-PCR was performed to assess the integrity of the RNA. First, cDNA was synthesized using 1 µg total RNA, the Transcriptor First Strand cDNA Synthesis Kit, and oligo(dT) primer (Roche). Real-time TaqMan PCR for β-actin (97 bp) was then performed on each sample using the Universal ProbeLibrary (UPL) probe (Roche) and the Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The UPL probe sequences were as follows: left, CCAACCGCGAGAAGATGA; right, CCAGAGGCGTACAGGGATAG. Cycle parameters were 95℃ for 10 minutes and 50 cycles of 10 seconds at 95℃ and 60 seconds at 58℃.
Nam S.K., Im J., Kwak Y., Han N., Nam K.H., Seo A.N, & Lee H.S. (2014). Effects of Fixation and Storage of Human Tissue Samples on Nucleic Acid Preservation. Korean Journal of Pathology, 48(1), 36-42.
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10

Quantitative Real-Time PCR Analysis

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Mid-log phase yeast cells were collected and used to determine the relative expression levels via qPCR. Total RNAs were isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s instructions. In total 1 µg of the RNA samples were then reversed transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit using oligo-dT primer (Roche, Indianapolis, IN, USA). The qPCR experiments were carried out using SYBR Green-based method in the QuantStudio 7 Flex Real-Time PCR System (ThermoFisher Scientific).
Lian J., HamediRad M., Hu S, & Zhao H. (2017). Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system. Nature Communications, 8, 1688.
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