Megaplex preamp primers

Manufactured by Thermo Fisher Scientific

Sourced in United States, Italy

Megaplex PreAmp Primers are a set of primers designed for pre-amplification of multiple target sequences prior to downstream analysis. The primers are optimized to provide efficient and unbiased amplification of target regions.

Automatically generated - may contain errors

Lab products found in correlation

44 protocols using megaplex preamp primers

Genome-Wide miRNA Profiling in FFPE Tissue

Cited 1 time

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

In our discovery phase we conducted genome-wide miRNA profiling using Taqman MicroRNA Arrays, also known as Taqman Low Density Array (TLDA) ‘Pool A’ Card version 3.0, due to their established use for miRNA expression analysis in FFPE tissue [28 (link),29 (link)]. This 384-microfluidic array was designed to perform quantitative reverse transcriptase (qRT-PCR) reactions simultaneously (Applied Biosystems, Austin, TX, USA) on 378 mature miRNAs (and 6 endogenous controls) that tend to be functionally defined. Using 20 nanograms (ng) of total RNA as input, cDNA was synthesized with highly multiplexed Megaplex RT primers, pre-amplified with Megaplex PreAmp Primers, mixed with TaqMan Universal PCR Master Mix (Applied Biosystems), and loaded onto TLDA cards for expression analysis.

Permuth-Wey J., Chen Y.A., Fisher K., McCarthy S., Qu X., Lloyd M.C., Kasprzak A., Fournier M., Williams V.L., Ghia K.M., Yoder S.J., Hall L., Georgeades C., Olaoye F., Husain K., Springett G.M., Chen D.T., Yeatman T., Centeno B.A., Klapman J., Coppola D, & Malafa M. (2015). A Genome-Wide Investigation of MicroRNA Expression Identifies Biologically-Meaningful MicroRNAs That Distinguish between High-Risk and Low-Risk Intraductal Papillary Mucinous Neoplasms of the Pancreas. PLoS ONE, 10(1), e0116869.

+ Open protocol

+ Expand

Plasma miRNA Profiling Using TaqMan Assays

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

TaqMan® Array Human MicroRNA A and B Cards v2.0 (Cat no 4400238, Applied Biosystems) were employed to examine the expression of 667 miRNAs in 48 plasma samples in the discovery cohort. Reverse transcription and quantitative PCR (qPCR) were performed on equal volumes of RNA from each sample according to the manufacturer’s instructions using TaqMan® MicroRNA Reverse Transcription Kit (Cat no 4366596, Applied Biosystems) and Megaplex RT Primers to convert the miRNAs to cDNA, TaqMan® PreAmp Master Mix (Cat no 4391128, Applied Biosystems) and Megaplex PreAmp Primers for a preamplification step before real-time analysis. qPCR was performed using TaqMan® Universal Master Mix II, no UNG (Cat no 4440048, Applied Biosystems) on the 7900HT Fast Real-Time PCR system (Applied Biosystems). The Sequence Detector System software version 2.2.2 was utilised to generate study files using a fixed threshold value of 0.1 for statistical analysis (accession no: GSE67075).

Aherne S.T., Madden S.F., Hughes D.J., Pardini B., Naccarati A., Levy M., Vodicka P., Neary P., Dowling P, & Clynes M. (2015). Circulating miRNAs miR-34a and miR-150 associated with colorectal cancer progression. BMC Cancer, 15, 329.

+ Open protocol

+ Expand

Profiling Liver microRNA Expression

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

Total RNA was isolated from transplanted livers using the
mirVana miRNA isolation kit (Applied Biosystems, Foster
City, CA). Normal non-transplanted DA and Lewis livers were used as controls and
calibrators for microarray data analysis. RNA samples from each group (n=3) of
transplanted or normal animals were prepared using TaqMan microRNA reverse
transcription kit and the corresponding Megaplex primer pools (Applied
Biosystems) as previously described¹¹. Pre-amplification of cDNA samples was performed with
TaqMan PreAmp master mix and corresponding Megaplex PreAmp primers (Applied
Biosystems). TaqMan array rodent microRNA A Card v2.0 (Applied Biosystems) were
used. Complete profiles from three livers in each group were analyzed. Liver
mononuclear cells were isolated as previously described¹². Data was analyzed using SDS
and RQ software (Applied Biosystems). Fold changes for each miRNA were
normalized to the endogenous control RNU6. The expression fold changes were
calculated using the comparative Ct method and compared to the relevant normal
tissue. qPCR was used to determine the relative expression of specific miRNA as
we have previously described⁶.

Vitalone M.J., Wai L., Fujiki M., Lau A.H., Littau E., Esquivel C., Martinez O.M, & Krams S.M. (2016). Liver microRNA profile of induced allograft tolerance. Transplantation, 100(4), 781-790.

+ Open protocol

+ Expand

Multiplex RT-PCR for miRNA Profiling

Cited 1 time

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

We used a fixed volume of 3 μl of total purified for the reverse transcription (RT). A set of 754 miRNAs were processed in CSF samples, using TaqMan miRNA Reverse Transcription Kit and Multiplex RT Assays (Human pool sets A and B) (Applied Biosystems) as previously described [18 (link)]. For RNA isolated from plasma samples, we used custom RT primer pool directed to the miRNAs identified as altered in CSF following the appropriate protocol. Each protocol has demonstrated sensitivity, specificity as well as repeatable and accurate results [19 (link)]. RT products for both CSF and plasma were then preamplified by means of TaqMan PreAmp Master Mix and/or Megaplex PreAmp Primers (Human pool sets A and B) (Applied Biosystems), or by custom preamplification primer pool, respectively. Preamplification step is mandatory before real-time PCR (RT-PCR) reaction when analytical sensitivity is the utmost importance and the sample is limiting [18 (link)].

Muñoz-San Martín M., Reverter G., Robles-Cedeño R., Buxò M., Ortega F.J., Gómez I., Tomàs-Roig J., Celarain N., Villar L.M., Perkal H., Fernández-Real J.M., Quintana E, & Ramió-Torrentà L. (2019). Analysis of miRNA signatures in CSF identifies upregulation of miR-21 and miR-146a/b in patients with multiple sclerosis and active lesions. Journal of Neuroinflammation, 16, 220.

+ Open protocol

+ Expand

High-throughput Profiling of Human miRNAome

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

Whole-miRNAome analsysis was performed with the TaqMan^® Array Human microRNA system (Applied Biosystems, USA) as per manufacturer’s protocols. This provided a high-throughput method to quantitatively screen the expression of 754 unique miRNAs, covering the broad majority of human miRNAs in the Sanger miRBase v14 miRNA database [44 (link)]. For this, cDNA prepared from the sample RNA underwent qRT-PCR on a pair of 384-well plates containing lyophilised primer/probe pairs. Three biological replicates were analysed for both functional and negative control samples. To enhance the detection of low-concentration miRNAs, a pre-amplification step was included using the TaqMan PreAmp Master Mix with the Megaplex PreAmp Primers (Applied Biosystems, USA). The manufacturer’s DataAssist 3.0 software was used to batch-analyse the expression results. MiRNAs that were deregulated with a P-value of ≤0.05 were included in the final dataset.

Vencken S.F., Sethupathy P., Blackshields G., Spillane C., Elbaruni S., Sheils O., Gallagher M.F, & O’Leary J.J. (2014). An integrated analysis of the SOX2 microRNA response program in human pluripotent and nullipotent stem cell lines. BMC Genomics, 15, 711.

+ Open protocol

+ Expand

TaqMan miRNA Array Analysis in HCAECs

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

miR Arrays were performed using TaqMan Array Human MicroRNA Card A v2.0 (Applied Biosystems Cat. # 4398965) in a 7900 HT fast real-time PCR instrument (Applied Biosystems). For cDNA preparation, 0.5 µg RNA was reversely transcribed by use of the TaqMan MicroRNA Reverse Transcription Kit and specific Megaplex RT Primers (Applied Biosystems, Cat. # 4399966). Subsequently, preamplification was performed using 2.5 µL of the RT product, Megaplex PreAmp Primers (Applied Biosystems, Cat. # 4399233), and the TaqMan PreAmp Master Mix (Applied Biosystems, Cat. # 4488593). 9 µL of the preamplification product was used for the final amplification with the MicroRNA Array Card and TaqMan Universal Master Mix II. The amplification curves were analysed with the Program RQ manager Version 1.2.1 (Applied Biosystems) to calculate CT values, using the same threshold across all respective HCAEC and EV samples. Only miRs that were stably expressed upon all three samples of control and hnRNPU knockdown HCAECs and EVs were included into the final analysis. ΔΔCT values were calculated by use of RNU-44, because RNU-44 was the most stably expressed endogenous control across the EV samples as assessed by CT value standard deviation: RNU-6b: SD 1.45, RNU-44: SD 0.79, RNU-48: SD 0.93.

Zietzer A., Hosen M.R., Wang H., Goody P.R., Sylvester M., Latz E., Nickenig G., Werner N, & Jansen F. (2020). The RNA-binding protein hnRNPU regulates the sorting of microRNA-30c-5p into large extracellular vesicles. Journal of Extracellular Vesicles, 9(1), 1786967.

+ Open protocol

+ Expand

MicroRNA Profiling of Radioresistant Cancers

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

Global microRNA expression profiling of FFPE samples in a discovering set comprised of 9 patients (5 from the radioresistant group and 4 from the radiosensitive group) was performed using the TaqMan Human MicroRNA Cards Set v2.0 (Applied Biosystems), allowing the evaluation of the expression level of 667 human microRNAs. Forty nanograms of total RNA from each sample was reverse transcribed into cDNA using the TaqMan miRNA Reverse Transcription Kit (Applied Biosystems) and Megaplex RT Primers (Applied Biosystems). Next, the product obtained from the RT reactions was pre-amplified using the TaqMan PreAmp Master Mix Kit (Applied Biosystems) and Megaplex PreAmp Primers (Applied Biosystems). The amplified-cDNA was then transferred to the TaqMan Human MicroRNA Cards Set v2.0 and the amplification was carried out in the 7900HT Real-Time PCR System (Applied Biosystems). The data obtained was analyzed using the software DataAssist v3.0 (Applied Biosystems). The fold-change difference between radioresistant and radiosensitive cases was calculated using the 2^−ΔΔCt method [23 (link)]. The small nuclear RNA U6 was used as endogenous control and the radiosensitive cases were assigned as reference. Cases were scored as differentially expressed if a 4-fold-change increase was observed.

Maia D., de Carvalho A.C., Horst M.A., Carvalho A.L., Scapulatempo-Neto C, & Vettore A.L. (2015). Expression of miR-296-5p as predictive marker for radiotherapy resistance in early-stage laryngeal carcinoma. Journal of Translational Medicine, 13, 262.

+ Open protocol

+ Expand

Whole Blood miRNA Extraction and Profiling

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

Peripheral whole blood from 530 participants was collected in Tri Reagent solution (Molecular Research Center, Cincinnati, OH) within 24 h of participant enrollment and stored at − 80 °C. Tri Reagent is a robust miRNA stabilization method for long-term storage and can generate reproducible results without degradation [16 (link)]. Total RNA containing small RNA was extracted from whole blood. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), and RNA integrity numbers (RIN) were reported. Total RNA with RIN of 6.5–10 was processed for complementary DNA synthesis using TaqMan Megaplex RT primer pools A or B and then amplified with Megaplex PreAmp primers (Applied Biosystems, Foster City, CA). One control sample in the validation cohort was excluded due to few detectable miRNAs.

Zhu Z., Liang L., Zhang R., Wei Y., Su L., Tejera P., Guo Y., Wang Z., Lu Q., Baccarelli A.A., Zhu X., Bajwa E.K., Taylor Thompson B., Shi G.P, & Christiani D.C. (2017). Whole blood microRNA markers are associated with acute respiratory distress syndrome. Intensive Care Medicine Experimental, 5, 38.

+ Open protocol

+ Expand

miRNA Profiling in Extracellular Vesicles

Cited 1 time

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

Ten nanograms of each RNA sample were reverse transcribed using Megaplex RT Primer Pools A and B (Applied Biosystems) and cDNA was pre-amplified using Megaplex PreAmp Primers (Applied Biosystems), according to manufacturer’s protocol. The pre-amplification reaction was set as 16 cycles. Samples were diluted, and real-time PCR was performed using TaqMan OpenArray MicroRNA Rodent Panels A and B and QuantStudio 12K Flex system (Applied Biosystems). The Ct value of each miR in each sample was normalized to average expression of all the miRs with valid Ct values in all samples (global normalization) [53 (link)]. Only Ct values < 28 showing amplification score higher than 1.0 and Ct confidence higher than 0.8 were accepted. For eGFP versus non-eGFP qPCR comparison analysis, miR was isolated from vEVs and as described. Ten nanograms of material was used for cDNA synthesis using TaqMan MicroRNA Reverse Transcription Kit (4366596, Applied Biosystems). Standard qPCR reactions were performed without pre-amplification using TaqMan Universal Master Mix II (444043, Applied Biosystems) and TaqMan MicroRNA Assay (001984, Applied Biosystems) according to manufacturer’s protocol. Ct values 35 or higher were considered undetectable.

Germano J.F., Sawaged S., Saadaeijahromi H., Andres A.M., Feuer R., Gottlieb R.A, & Sin J. (2019). Coxsackievirus B infection induces the extracellular release of miR-590-5p, a proviral microRNA. Virology, 529, 169-176.

+ Open protocol

+ Expand

Profiling Oral Tongue SCC Lymph Node MicroRNAs

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

In the ‘discovery set’, a global microRNA expression profiling was performed in lymph nodes from six FFPE oral tongue SCC samples (four with macrometastases and two with non-metastatic nodes) using TaqMan Human MicroRNA Arrays (Applied Biosystems, Foster City, CA, USA).
Total RNA (40 ng) from each lymph node sample were reverse transcribed into cDNA using the TaqMan microRNA Kit and Megaplex RT Primers (both from Applied Biosystems). After synthesis, the cDNA was pre-amplified using the TaqMan PreAmp Master Mix Kit and Megaplex PreAmp Primers (Applied Biosystems). The amplified-cDNA was then transferred to the TaqMan Human MicroRNA Array plates and the amplification was carried out in an Applied Biosystems 7900HT Real-Time PCR system.
The data obtained was analyzed using the software DataAssist v3.0 (Applied Biosystems). The fold-change difference between metastatic and non-metastatic lymph node samples was calculated using the 2^-ΔΔCt method [39 (link)]. The small nuclear RNA U6 was used as an endogenous control and the non-metastatic group was assigned as a reference since this was the most stable control in the assay.

de Carvalho A.C., Scapulatempo-Neto C., Maia D.C., Evangelista A.F., Morini M.A., Carvalho A.L, & Vettore A.L. (2015). Accuracy of microRNAs as markers for the detection of neck lymph node metastases in patients with head and neck squamous cell carcinoma. BMC Medicine, 13, 108.

+ 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!