Rt2 sybr green pcr kit

Manufactured by Qiagen

Sourced in United States

The RT2 SYBR Green PCR kit is a real-time PCR assay designed for gene expression analysis. It includes reagents for efficient reverse transcription and SYBR Green-based quantitative PCR. The kit allows for accurate and sensitive detection of target gene expression.

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Lab products found in correlation

Miscript sybr green pcr kit, by Qiagen (4 mentions) Rt2 first strand kit, by Qiagen (2 mentions) Rotor gene q system, by Qiagen (2 mentions) Rotor gene q real time pcr system, by Qiagen (2 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Mirneasy serum plasma extraction kit, by Qiagen (1 mentions) Cfx96 real time system, by Bio-Rad (1 mentions) Miscript 2 rt kit, by Qiagen (1 mentions) Mirneasy kit, by Qiagen (1 mentions)

Close spelling variants of this product

RT2 SYBR Green SYBR Green kit SYBR Green

8 protocols using rt2 sybr green pcr kit

Quantitative Analysis of lncRNA CASC2 and miR-21-5p

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These reactions were achieved using RT2 SYBR Green PCR kit (Qiagen, Maryland, USA) for LncRNA CASC2 expression while miScript SYBR Green PCR kit (Qiagen, Valencia, CA, USA) for the detection of miRNA-21-5p.
The LncRNA CASC2 RefSeq Accession number was NR_026939. GAPDH served as an internal control for measuring CASC2 expression level [30 (link)].
Moreover, the miR-21-5p catalog number was MS0009079. SNORD 68 was used as an internal reference for miR-21-5p. After analysis of the data using the quantification of the cycle threshold (CT), relative expression of LncRNA CASC2 and miR-21-5p was calculated using the Eq. 2^−ΔΔCt. Fold change (FC) values less than 1 indicate downregulation, while fold change values more than 1 indicate upregulation of noncoding RNAs [31 (link), 32 (link)]. Control FC values were put as 1.

Ayoub S.E., Shaker O.G., Masoud M., Hassan E.A., Ezzat E.M., Ahmed M.I., Ahmed R.I., Amin A.A., Abd El Reheem F., Khalefa A.A, & Mahmoud R.H. (2024). Altered expression of serum lncRNA CASC2 and miRNA-21-5p in COVID-19 patients. Human Genomics, 18, 18.

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Serum lncRNA Quantification Protocol

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Materials used to execute this work include; one- MiRNeasy Serum/Plasma extraction kit (Qiagen, Valencia, CA, USA) was used for total RNA extraction from serum. Two- The RT2 First Strand Kit (Qiagen, Valencia, CA, USA) was used for reverse transcription to produce cDNA. Three- The RT2 SYBR Green PCR kit (Qiagen, Maryland, USA) was used to perform qRT-PCR along with specific primers supplied by Qiagen, (Valencia, CA, USA); for IFNG-AS1 (Catalog no; 330701LPH20079A, Accession no, ENST00000536914.0) and GAS5 (Catalog no; 330701LPH11340A, Accession no, NR_002578.2) and we used GAPDH primer (Catalog no: 330701 LPH31725A, Accession no: ENST00000496049.0) to standardize the expression pattern and quantify the target long non-coding RNAs.

Ali M.A., Hussein S.K., Khalifa A.A., El Amin Ali A.M., Farhan M.S., Ibrahim Amin A.A, & Mohamed E.A. (2022). The Ifng antisense RNA 1 (IFNG-AS1) and growth arrest-specific transcript 5 (GAS5) are novel diagnostic and prognostic markers involved in childhood ITP. Frontiers in Molecular Biosciences, 9, 1007347.

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Quantification of HOTTIP and miR-615-3p Expression

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RT-qPCR of the HOTTIP expression was performed using the RT2 SYBR Green PCR kit (Qiagen, Maryland, MY, USA). The expression of miR-615-3p was quantified using the miScript SYBR Green PCR kit (Qiagen, Valenica, CA, USA) following the instructions of the manufacturer. The RefSeq accession no. of HOTTIP was NR_037843.3, and the catalog number of miR-615-3p was MS00029225. All the reactions were done in the Rotor gene Q System (Qiagen) on a 20-μL reaction mixture with the following settings for the HOTTIP assessment: 95 °C for 10 min; subsequently, 40 cycles at 95 °C for 15 s and 60 °C for 60 s. However, for the assessment of miR-615-3p, the cycling conditions were as follows: 95 °C for 30 min, followed by 40 cycles at 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s.
The expression values of HOTTIP were normalized using GAPDH as an endogenous reference gene [17 (link),18 (link)]. However, for the calculation of the miR-615-3p expression level, SNORD 68 was used as an internal control. The GAPDH primer sequences were forward 5′-CCCTTCATTGACCTCAACTA-3′ and reverse 5′-TGGAAGATGGTGATGGGATT-3′. The catalog number of SNORD 68 was MS00033712. The equation 2^−ΔΔCt was used to calculate the fold changes (FC) of HOTTIP and miR-615-3p [19 (link)]. The FC of the healthy group was assumed as 1.

Abdelaleem O.O., Shaker O.G., AbdelHafez M.N., Abdelghaffar N.K., Eid H.M., Zaidan M., Khalefa A.A., Ahmed N.A., Hemeda N.F., Zaki O.M., Awaji A.A, & Mohammed S.R. (2021). The Influence of rs1859168 Polymorphism on Serum Expression of HOTTIP and Its Target miR-615-3p in Egyptian Patients with Breast Cancer. Biomolecules, 11(5), 733.

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Quantitative Analysis of Serum lncRNAs

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Previously, target lncRNAs in serum were measured [[22] , [23] (link), [24] (link)]. The levels of the lncRNAs NEAT1, HOTAIR, and GAS5 were determined using quantitative real-time PCR (RT-qPCR). RT-qPCR was performed using the Rotor-gene Q real-time PCR system (Qiagen, USA). We used the RT2 SYBR Green PCR kit (Qiagen, Germantown, MD, USA), a predesigned specific primer for each lncRNA, and the housekeeping gene (GAPDH) [36 (link)] were obtained from (Qiagen, Valencia, CA, USA), NEAT1 (Catalog no: 330701 LPH15809A, Accession no: NR_028272.1), GAS5 (Catalog no; 330701LPH11340A, Accession no, NR_002578.2), HOTAIR (Catalog no; 330701LPH07360A Accession no; NR_003716.3), and GAPDH housekeeping gene (Catalog no: 330701 LPH31725A, Accession no: ENST00000496049.0) to execute the PCR reactions. The PCR cycling procedure for quantifying lncRNAs begins with a 10-min incubation at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. The 2^−ΔΔCt equation was used to calculate the serum fold changes of NEAT1, GAS5, and HOTAIR. Non-coding RNAs with a fold change (FC) less than one were downregulated, whereas those with an FC more than one were upregulated [25 (link)]. The controls FC values were set as one.

Ali M.A., Shaker O.G., Khalifa A.A., Ezzat E.M., Elghobary H.A., Abdel Mawla T.S., Elkhateeb A.F., Elebiary A.M, & Elamir A.M. (2022). LncRNAs NEAT1, HOTAIR, and GAS5 expression in hypertensive and non-hypertensive associated cerebrovascular stroke patients, and its link to clinical characteristics and severity score of the disease. Non-coding RNA Research, 8(1), 96-108.

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Quantitative Analysis of miRNA and mRNA

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Total RNA containing miRNA was isolated by using a miRNeasy kit (Qiagen, Germantown, CA, USA) according to the manufacturer’s protocol. The purity of the total RNA was assessed using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) with criteria of purity met by A260/280 ratios of 1.8–2.1. Total RNA was then converted into cDNA and micDNA by using a RT2 First Strand kit and a miScript II RT kit (Qiagen, Germantown, CA, USA), respectively. Quantitative RT-PCR (qRT-PCR) was performed using an RT2 SYBR green PCR kit (Qiagen, Germantown, CA, USA) for mRNA and a miScript SYBR green PCR kit (Qiagen) for miRNA, using a CFX96 Real-Time System (BioRad, Hercules, CA, USA). We purchased primers for miR-200c-3p (catalog no. MS00003752) and the reference gene, RNU6 (catalog no. MS00033740), and primers for mRNA DLC1 (catalog no. PPH00438B), CDH1 (catalog no. PPH00135F), and the reference gene PPIA (catalog no. PPH01319G) from Qiagen. The relative expression was calculated using the 2-ΔΔCT method.

Ankasha S.J., Shafiee M.N., Abdul Wahab N., Raja Ali R.A, & Mokhtar N.M. (2021). Oncogenic Role of miR-200c-3p in High-Grade Serous Ovarian Cancer Progression via Targeting the 3′-Untranslated Region of DLC1. International Journal of Environmental Research and Public Health, 18(11), 5741.

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Quantification of Serum lncRNAs by qRT-PCR

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It has previously been shown that lncRNAs were expressed in serum (Mohammed et al., 2022 (link))– (Visconti et al., 2020 (link)). The RT2 SYBR Green PCR kit (Qiagen, Maryland, USA) was used to evaluate the expression of the lncRNAs IFNG-AS1 and GAS5 in serum using specific primers supplied by Qiagen, (Valencia, CA, USA); for IFNG-AS1 (Catalog no; 330701LPH20079A, Accession no, ENST00000536914.0) and GAS5 (Catalog no; 330701LPH11340A, Accession no, NR_002578.2) and we used GAPDH primer (Catalog no: 330701 LPH31725A, Accession no: ENST00000496049.0) to standardize the expression pattern and quantify the target long non-coding RNAs (Li et al., 2016 (link)), (Peng et al., 2021 (link)). The reaction mix was prepared in a nuclease-free tube according to the manufacturer’s protocol for a 25 μl per well reaction volume. The Rotor-gene Q Real-time PCR system (Qiagen, USA) was used to perform quantitative real-time PCR under the following conditions: 95°C for 10 min, then 45 cycles of 15s at 95 and 60°C for 1 min.

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Quantitative Analysis of GAS5 and miR-137 Expression

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Assessment of GAS5 was evaluated by the use of RT2 SYBR Green PCR kit (Qiagen, Maryland, USA). However, the miScript SYBR Green PCR kit (Qiagen, Valenica, CA, USA) was used in the assessment of miR-137 according to the kit instructions with the aid of Rotor gene Q System (Qiagen) on 20 μl reaction mixture.
The RefSeq Accession no. of GAS5 was NR_002578.2 and the catalog number of miR-137 was MS00003486. Assessment of GAS5 was done per the following cycling steps: 95 °C for 10 min, afterward, 40 cycles at 95 °C for 15 s and 60 °C for 60 s. On the other hand, assessment of miR-137 was performed according to the subsequent cycling steps: 95 °C for 30 min, subsequently, 40 cycles at 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s.
GAS5 expression values were normalized by the use of GAPDH as an internal control^{44 (link),45 (link)}. Though, miR-137 expression values were normalized using SNORD 68 as an internal reference gene. The GAPDH primer sequences were forward, 5′-CCCTTCATTGACCTCAACTA-3′, and reverse 5′-TGGAAGATGGTGATGGGATT-3′. The catalog number of SNORD 68 was MS00033712. Calculation of the fold change (FC) of GAS5 and miR-137 was done with the aid of the equation 2^−ΔΔCt^{46 (link)}. The FC of control individuals was assumed as 1.

Mahmoud R.H., Hefzy E.M., Shaker O.G., Ahmed T.I., Abdelghaffar N.K., Hassan E.A., Ibrahim A.A., Ali D.Y., Mohamed M.M, & Abdelaleem O.O. (2021). GAS5 rs2067079 and miR-137 rs1625579 functional SNPs and risk of chronic hepatitis B virus infection among Egyptian patients. Scientific Reports, 11, 20014.

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Quantification of lncRNA Serum Expression

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In the reverse transcription (RT) step, 60 ng of RNA was used. GAPDH (internal control), readymade primers, and RT2 SYBR Green PCR kit (Qiagen, Germantown, MD, USA) were used to assess lncRNA serum expression (Duan et al., 2016 (link)). The RT2 lncRNA PCR assay for MALAT1 was dependent on predesigned primers catalog no: 330701 LPH18065A, Accession no: NR_002819.2. The RT2 lncRNA PCR assay for NEAT1 was dependent on predesigned primers catalog no: 330,701 LPH15809A, Accession no: NR_028272.1. We used GAPDH as an internal housekeeping gene (Adriaens et al., 2016) (Catalog no: 330,701 LPH31725A, Accession no: ENST00000496049.0). Twenty microliter reaction mixtures were used in RT-PCR by mixing 10 µl of master mix, 1 µl readymade assay primer, 2.5 µl of dil. cDNA, and 5.5 µl RNAase-free water. PCR conditions were as follows: 95 °C for 10 min, after that, 45 cycles at 95 °C for 15 s and 60 °C for 60 s. Gene expression relative to internal control (2^−ΔCt) was determined. A melt curve analysis was done to ensure the specificity of the corresponding RT-PCR reactions. Fold change was calculated using 2^−ΔΔCt for relative quantification (Livak and Schmittgen, 2001 (link)). For the control sample, ΔΔCt equals zero, and 2⁰ is equal to one (Shaker et al., 2019a (link), Shaker et al., 2019b (link)).

Mohammed A., Shaker O.G., Khalil M.A., Elsabagh Y.A., Gomaa M., Ahmed A.M, & Erfan R. (2022). Association of long non-coding RNAs NEAT1, and MALAT1 expression and pathogenesis of Behçet's disease among Egyptian patients. Saudi Journal of Biological Sciences, 29(8), 103344.

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