Quantitec reverse transcription kit

Manufactured by Qiagen

Sourced in Germany, United States, Canada, Italy

The QuantiTec Reverse Transcription Kit is a reagent system designed for the conversion of RNA to cDNA. It enables the synthesis of first-strand cDNA from total RNA or poly A+ RNA templates.

Automatically generated - may contain errors

Lab products found in correlation

78 protocols using quantitec reverse transcription kit

Analyzing Honeybee Eye Photoreceptors

Check if the same lab product or an alternative is used in the 5 most similar protocols

We removed both eyes (the retina and a small part of the optic lobe) of workers since the different photoreceptor types seem to be approximately evenly distributed in the compound eye [21 (link)]. In contrast, drones lack green photoreceptor cells in the dorsal part of their eye [17 (link), 22 (link), 23 (link)], and we thus separated the dorsal from the ventral part. However, the ventral worker-like part of the retina covers only ca. 20% of the entire eye [22 (link), 23 (link)], and we were unable to clearly dissect this part of the eye without contamination and thus used only the dorsal part in drones for further analysis. The eyes were dissected on ice and remained frozen until they were transferred into PCR tubes for further RNA extraction. To isolate mRNA, we used TRIzol Reagent (5 Prime, Hilden, Germany) and the Gold HP total RNA Kit (Peqlab Biotechnologie, Erlangen, Germany) following the manufacturer’s protocol. Quantity and quality of mRNA was estimated by spectrophotometric measurement and gel-electrophorese (1% agarose gel, stained with Midori Green Direct; Nippon Genetics Europe, Dueren, Germany). cDNA synthesis was performed by means of QuantiTec Reverse Transcription Kit (Quiagen, Hilden, Germany) according to the manufacturer’s protocol. All cDNAs were stored at -20C° until used for quantitative real-time PCR (qPCR).

Lichtenstein L., Grübel K, & Spaethe J. (2018). Opsin expression patterns coincide with photoreceptor development during pupal development in the honey bee, Apis mellifera. BMC Developmental Biology, 18, 1.

+ Open protocol

+ Expand

RT-qPCR Gene Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

RNA was prepared with the RNeasy Mini Kit (Qiagen). To obtain cDNA, the QuantiTec Reverse Transcription Kit (Quiagen) was used. RT-qPCR was performed using the LightCycler 480 Probes Master Mix (Roche) according to the manufacturer´s protocol and a LightCycler 480 II (Roche Diagnostics). Primers and probes were designed with the Universal Probe Library Assay Design Center provided by Roche Diagnostics. RNAPol2 and YWHAZ were used as reference genes. Complete primer sequences and probes are presented in Supplementary Table 1.

Kuhn L.B., Valentin S., Stojanovic K., Strobl D.C., Babushku T., Wang Y., Rambold U., Scheffler L., Grath S., John-Robbert D., Blum H., Feuchtinger A., Blutke A., Weih F., Kitamura D., Rad R., Strobl L.J, & Zimber-Strobl U. (2022). RelB contributes to the survival, migration and lymphomagenesis of B cells with constitutively active CD40 signaling. Frontiers in Immunology, 13, 913275.

+ Open protocol

+ Expand

Validating RNA-seq with qRT-PCR

Check if the same lab product or an alternative is used in the 5 most similar protocols

Result obtained in the RNA-seq analysis were validated by quantitative reverse transcription PCR (qRT-PCR) of 20 selected genes, which represented differentially and non-differentially expressed genes in the presence of apigenin and salt. Total RNA was isolated using a High Pure RNA Isolation Kit (Roche) and RNAase Free DNA Set (Qiagen), according to the manufacturer’s instructions. This (DNA-free) RNA was reverse transcribed into cDNA using a QuantiTec Reverse Transcription Kit (Qiagen). Quantitative PCR was performed using a LightCycler 480 (Roche) with the following conditions: 95 °C, 10 min; 95 °C, 30 s; 50 °C, 30 s; 72 °C, 20 s; forty cycles, followed by the melting curve profile from 60 to 95 °C to verify the specificity of the reaction. The R. tropici CIAT 899 16S rRNA gene was used as an internal control to normalize gene expression. The fold-changes of two biological samples with three technical replicates of each condition were obtained using the ∆∆C_t method [38 (link)]. Selected genes and primers are listed in Additional file 2.

Pérez-Montaño F., del Cerro P., Jiménez-Guerrero I., López-Baena F.J., Cubo M.T., Hungria M., Megías M, & Ollero F.J. (2016). RNA-seq analysis of the Rhizobium tropici CIAT 899 transcriptome shows similarities in the activation patterns of symbiotic genes in the presence of apigenin and salt. BMC Genomics, 17, 198.

+ Open protocol

+ Expand

RNA Extraction and RT-qPCR Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

RNA was extracted from tumourspheres or cell cultures using RNeasy micro kit (Qiagen, Chadstone Centre, VIC, Australia). cDNA was synthesized with the QuantiTec Reverse transcription kit (Qiagen) and Real-time quantitative PCR was performed in an ABI Prism 7500 Sequence Detection System using the thermocycler conditions recommended (Applied Biosystems, Grand Island, NY, USA). Detection was performed with SYBR Green PCR Master Mix and analysis with the ΔCt method. Oligonucleotides used can be seen on Supplementary Table S1.

Vega F.M., Colmenero-Repiso A., Gómez-Muñoz M.A., Rodríguez-Prieto I., Aguilar-Morante D., Ramírez G., Márquez C., Cabello R, & Pardal R. (2019). CD44-high neural crest stem-like cells are associated with tumour aggressiveness and poor survival in neuroblastoma tumours. EBioMedicine, 49, 82-95.

+ Open protocol

+ Expand

Quantitative Real-Time PCR Analysis

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total RNA was extracted from Tet-21/N cells and reverse transcription reaction was performed using Quantitec Reverse Transcription Kit (Qiagen, Hilden, Germany). cDNA thus obtained was analyzed by qPCR using SYBR Green 2X PCR Master Mix (Applied Biosystem, Waltham, MA, USA). Each sample was run in triplicate and the expression of housekeeping beta-glucoronidase (GUSb) gene used for normalization as described.^{41 (link)} Primers are presented in Supplementary Table S2.

Ambrosio S., Saccà C.D., Amente S., Paladino S., Lania L, & Majello B. (2017). Lysine-specific demethylase LSD1 regulates autophagy in neuroblastoma through SESN2-dependent pathway. Oncogene, 36(48), 6701-6711.

+ Open protocol

+ Expand

Osteoclastogenesis Regulation by GDF11

Check if the same lab product or an alternative is used in the 5 most similar protocols

Expression levels of messenger RNA were measured by microarray analysis of mouse BMMs 24 h after stimulation in the presence of 100 ng ml⁻¹ M-CSF and 50 ng ml⁻¹ RANKL with or without 100 ng ml⁻¹ rGDF11. Total RNA was isolated using an RNeasy kit (Qiagen) according to the manufacturer's protocol. Microarray experiments were performed in triplicate using the Affymetrix Mouse Gene 1.0 ST Array. The robust multichip average method was used to normalize the gene expression raw data. We calculated the gene expression levels using Affymetrix Expression Console and Transcriptome Analysis Console 3.0 software. The threshold for differentially regulated transcripts was set as the fold change of 1.5 with a P value of <0.01.
For quantitative RT-PCR, cDNA was prepared from 2 μg RNA using a QuantiTec reverse transcription kit (Qiagen) and analysed with SYBR GreenMaster Mix (SABiosciences) in ABI7500 real-time PCR system (Applied Biosystems, Foster City, CA). The primers designed for each targeted gene are listed in Supplementary table 1. Relative expression was calculated using a 2^−ΔΔCt method by normalizing with Gapdh housekeeping gene expression and presented as fold increase relative to control.

Liu W., Zhou L., Zhou C., Zhang S., Jing J., Xie L., Sun N., Duan X., Jing W., Liang X., Zhao H., Ye L., Chen Q, & Yuan Q. (2016). GDF11 decreases bone mass by stimulating osteoclastogenesis and inhibiting osteoblast differentiation. Nature Communications, 7, 12794.

+ Open protocol

+ Expand

Quantitative Real-time PCR Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

RNA was extracted from MCF10A-AsiSIER cells using EuroGold Trifast (EuroClone). cDNA was generated using Quantitec Reverse Transcription Kit (Qiagen), according to manufacturer's protocol. Quantitative analysis was performed using SYBR Green 2X PCR Master Mix (Applied Biosystem). Each sample was run in triplicate and normalized to the expression of housekeeping beta-glucoronidase (GUS) gene as previously described [44 (link)]. Primers are presented in Supplementary Table S2.

Ambrosio S., Di Palo G., Napolitano G., Amente S., Dellino G.I., Faretta M., Pelicci P.G., Lania L, & Majello B. (2015). Cell cycle-dependent resolution of DNA double-strand breaks. Oncotarget, 7(4), 4949-4960.

+ Open protocol

+ Expand

RNA Isolation and qRT-PCR Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

RNA was isolated (Quick RNA Micro Prep kit Zymo Research) and reversely transcribed to cDNA (Quantitec-Reverse transcription kit, QIAgen) as per manufacturer’s instructions. Real time Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed by using BIO-RAD (CFX96 Touch) real time PCR detection system and SYBR Green master mix (Thermofisher). Expression of gene of interest was normalized to either 18S or 36B4 (Supplementary Table 1).

Afreen S., Bohler S., Müller A., Demmerath E.M., Weiss J.M., Jutzi J.S., Schachtrup K., Kunze M, & Erlacher M. (2020). BCL-XL expression is essential for human erythropoiesis and engraftment of hematopoietic stem cells. Cell Death & Disease, 11(1), 8.

+ Open protocol

+ Expand

Quantitative Gene Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

RNA was isolated by a Quick RNA Micro Prep kit (Zymo Research) and reversely transcribed to cDNA (Quantitec- Reverse transcription kit, QIAgen). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed by using BIO-RAD (CFX96 Touch) a RT-PCR detection system and SYBR Green master mix (Thermofisher). Expression of the gene of interest was normalized to the expression of either 18S or 36B4.

Bohler S., Afreen S., Fernandez-Orth J., Demmerath E.M., Molnar C., Wu Y., Weiss J.M., Mittapalli V.R., Konstantinidis L., Schmal H., Kunze M, & Erlacher M. (2020). Inhibition of the anti-apoptotic protein MCL-1 severely suppresses human hematopoiesis. Haematologica, 106(12), 3136-3148.

+ Open protocol

+ Expand

Quantitative Gene Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

RNA was extracted from Tet-21/N cells using EuroGold Trifast (EuroClone). cDNA was generated using Quantitec Reverse Transcription Kit (Qiagen), according to manufacturer's protocol. Quantitative analysis was performed using SYBR Green 2X PCR Master Mix (Applied Biosystem). Each sample was run in triplicate and normalized to the expression of housekeeping beta-glucoronidase (GUS) gene as previously described (34). Primers presented in Supplementary Table 1

Amente S., Milazzo G., Sorrentino M.C., Ambrosio S., Di Palo G., Lania L., Perini G, & Majello B. (2015). Lysine-specific demethylase (LSD1/KDM1A) and MYCN cooperatively repress tumor suppressor genes in neuroblastoma. Oncotarget, 6(16), 14572-14583.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!