miScript miRNA PCR Arrays

For SYBR® Green-based, real-time PCR profiling of miRNAs using the miScript PCR System

  • Sensitive and specific mature miRNA expression profiling
  • PCR arrays for pathway, disease, or miRNome profiling
  • Free online data analysis tools
miScript miRNA PCR Arrays are mature miRNA-specific forward primers (miScript Primer Assays) that have been arrayed in biologically relevant pathway-focused, disease-focused, or whole miRNome panels. miScript miRNA PCR Arrays are part of the miScript PCR System for miRNA detection and quantification. cDNA is prepared using the miScript II RT Kit with miScript HiSpec Buffer and is used as a template in real-time PCR with a miScript miRNA PCR Array and the miScript SYBR Green PCR Kit.

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miScript miRNA PCR Arrays 适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


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Pathway-Focused or miRNome miScript miRNA PCR Array layout for plate formats A, C, D, F, M.|miRNome miScript miRNA PCR Array and miScript miRNA HC PCR Array layout for plate formats E, G.|Pathway-Focused miScript miRNA PCR Array layout for plate formats E, G.|miScript miRNA PCR Array layout for Rotor-Disc format R.|miScript miRNA QC PCR Array layout for plate formats A, C, D, F.|miScript miRNA QC PCR Array layout for plate formats E and G.|miScript miRNA QC PCR Array layout for Rotor-Disc format R.|Whole miRNome profiling.|Analysis of expression of circulating miRNAs in serum or plasma holds huge potential as a source of new biomarkers.|
Wells A1 to G12 (1–84) each contain a miScript Primer Assay for a pathway/disease/functionally related mature miRNA. Wells H1 and H2 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells H3 to H8 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells H9 and H10 contain replicate reverse transcription controls (miRTC). Wells H11 and H12 contain replicate positive PCR controls (PPC).|Wells A1 to P12 (1–372) each contain a miScript Primer Assay for a pathway/disease/ functionally related mature miRNA. Wells P13 and P14 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells P15 to P20 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells P21 and P22 contain replicate miRTC miScript Primer Assays (miRTC). Wells P23 and P24 contain replicate positive PCR controls (PPC).|Pathway-Focused miScript miRNA PCR Arrays in formats E and G include 4 replicates of the same assays as provided in the 96-well formats A, C, D, and F.|Wells 1 to 84 each contain a miScript Primer Assay for a pathway/disease/functionally related gene. Wells 85 and 86 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells 87 to 92 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells 93 and 94 contain replicate miRTC miScript Primer Assays (miRTC). Wells 95 and 96 contain replicate positive PCR controls (PPC). Wells 97–100 are empty.|Wells 1 and 2 of each row contain replicate C. elegans miR-39 miScript Primer Assays (39). Well 3 of each row contains a miR-16 miScript Primer Assay (16). Well 4 of each row contains a miR-21 miScript Primer Assay (21). Well 5 of each row contains a miR-191 miScript Primer Assay (191). Wells 6 to 8 of each row each contain an assay for a different snoRNA (61 = SNORD61 assay, 95 = SNORD95 assay, 96A = SNORD96A assay). Wells 9 and 10 of each row contain replicate miRTC miScript Primer Assays (miRTC). Wells 11 and 12 of each row contain replicate positive PCR controls (PPC). These formats enable the quality assessment of up to 8 cDNA samples.|Wells 1 to 4 of each row contain replicate C. elegans miR-39 miScript Primer Assays (39). Wells 5 and 6 of each row contain replicate miR-16 miScript Primer Assays (16). Wells 7 and 8 of each row contain replicate miR-21 miScript Primer Assays (21). Wells 9 and 10 of each row contain replicate miR-191 miScript Primer Assays (191). Wells 11 to 16 of each row contain assays for different snoRNAs (61 = SNORD61 assay, 95 = SNORD95 assay, 96A = SNORD96A assay). Wells 17 to 20 of each row contain replicate miRTC miScript Primer Assays (miRTC). Wells 21 to 24 of each row contain replicate positive PCR controls (PPC). These formats enable the quality assessment of up to 32 cDNA samples.|Wells 1 and 2 contain replicate C. elegans miR-39 miScript Primer Assays. Well 3 contains a miR-16 miScript Primer Assay. Well 4 contains a miR-21 miScript Primer Assay. Well 5 contains a miR-191 miScript Primer Assay. Wells 6 to 8 each contain an assay for a different snoRNA (Well 6 = SNORD61 assay, Well 7 = SNORD95 assay, Well 8 = SNORD96A assay). Wells 9 and 10 contain replicate miRTC miScript Primer Assays (miRTC). Wells 11 and 12 contain replicate positive PCR controls (PPC). This pattern is repeated 7 additional times from wells 13 to 96. Wells 97 to 100 are empty. This format enables the quality assessment of up to 8 cDNA samples.|Total RNA was isolated from HCT 116 colorectal cancer cells that had been treated with ± 5-aza-2'-deoxycytidine (5-aza-2'-dC) demethylation reagent. This reagent irreversibly inhibits DNA methyltransferase driven methylation reactions by incorporating into DNA and covalently binding to the active site of the DNA methyltransferase. cDNA was prepared with miScript HiSpec Buffer using the miScript II RT Kit. The Human miRNome miScript miRNA PCR Array, in combination with the miScript SYBR® Green PCR Kit, was used to profile mature miRNA expression by real-time PCR. Data analysis was performed using the online miScript miRNA PCR Array Data Analysis Tool. [A] A scatter plot of 2-ΔCT values demonstrates that significant differences exist in the mature miRNA expression levels of the 2 samples. [B] A volcano plot shows regulated miRNAs (±4-fold is used as a cutoff and indicated by red lines). 104 miRNAs were strongly upregulated and 30 were strongly downregulated in 5-aza-2'-dC treated HCT 116 cells. When a p value of 0.05 is applied (indicated by blue line), the upregulation of 89 of the 104 miRNAs is significant, and the downregulation of 21 of the 30 miRNAs is significant.|Scatter plots show the relative miRNA expression levels from pooled RNA from healthy and colon cancer serum samples, characterized using miScript miRNA PCR Arrays.|
Performance
Discover biomarkers in serum or plasma samples

The range of pathway-focused miScript miRNA PCR Arrays is continually expanding to enable new discoveries about the roles of miRNAs in biological processes. The content of miScript miRNA PCR Arrays is selected using our proprietary methodology, which ensures that each array is up-to-date and biologically relevant. One of the most exciting areas of current miRNA research involves the assessment of miRNAs present in serum or plasma samples. The discovery of relatively stable, extracellular miRNAs in serum and plasma has generated huge interest in the use of changes in these miRNA levels as potential noninvasive biomarkers for a variety of diseases. As a result, the Serum & Plasma miScript miRNA PCR Array has been developed. This array enables rapid profiling of the 84 most relevant miRNAs associated with serum and plasma (see figure "Analysis of expression of circulating miRNAs in serum or plasma").

Profile entire miRNomes with a single cDNA sample

In addition to pathway-focused arrays, up-to-date miRNome miScript miRNA PCR Arrays are available for a variety of species including human, mouse, rat, dog, and rhesus macaque. With less than 1 µg RNA, the entire human miRNome can be rapidly profiled (see figure "Whole miRNome profiling"). Each assay in a miScript miRNA PCR Array has been bench verified to ensure sensitive and specific detection of mature miRNA by real-time PCR.

Principle

The miScript PCR System enables sensitive, specific miRNA quantification and profiling using SYBR Green real-time PCR. The miScript PCR System covers all the steps involved in conversion of RNA to cDNA and subsequent real-time PCR detection of miRNAs.

Mature miRNA profiling with the miScript PCR System uses the following components:

miScript II RT Kit — this kit enables simple, single-step cDNA synthesis. A single cDNA synthesis reaction can be used for detection of hundreds of miRNAs. The dual buffer system meets the distinctive needs of miRNA quantification using real-time PCR.
miScript PreAMP PCR Kit and Primer Mixes — this kit enables an optional preamplification step when working with limited RNA amounts.
miScript SYBR Green PCR Kit — this kit includes QuantiTect SYBR Green PCR Master Mix and the miScript Universal Primer, a reverse primer that allows detection of miRNAs in combination with a miScript Primer Assay or miScript miRNA PCR Array.
miScript miRNA PCR Array — Cutting-edge miScript miRNA PCR Arrays consist of mature miRNA-specific forward primers (miScript Primer Assays) arrayed in biologically relevant pathway- or disease-focused panels or miRNome panels. 
Procedure

The miScript miRNA PCR Array Service includes:

Total RNA (including miRNA) purificationPurification of total RNA from cells, tissues, blood, formalin-fixed paraffin-embedded (FFPE) samples, laser capture microdissection (LCM) samples, fine needle aspiration biopsies (FNAB), and other small samples if required Reverse transcription using the miScript II RT KitmiRNA profiling using a pathway-focused, disease-focused, or whole miRNome miScript miRNA PCR ArrayData analysis

Sample processing

For instructions regarding sample submission and shipping, contact QIAGEN Technical Services. Samples accepted are total RNA, cells, or tissue. An RNA Isolation Service is provided if required.

Before shipping:

Obtain a sample submission form from QIAGEN Technical Services. Complete the form and include it with the sample shipment. Include the amount of material and its storage conditions. Sign and date the form. Ship according to the instructions for your sample type.

Submitting cells or tissue samples

All sample collection should be carried out in a timely manner in an RNase free environment. Pay close attention to the minimum sample amounts required. If you have less than these minimum amounts, or your previously isolated samples have been stored differently then we recommend, contact QIAGEN Technical Services before submitting your samples.

Cells:

Provide at least 1 million (106) cells for each sample. Specify the number of cells on the order form. Snap freeze the cell pellets in liquid nitrogen. Alternatively, provide homogenized cell lysates (see the miRNeasy Mini Handbook for details of lysis using QIAzol Lysis Reagent) Store samples frozen at -80 ºC, and ship on dry ice.

Tissue:

If possible, provide at least 50 mg of tissue for each sample. If possible, specify the weight of the tissue on the order form. Upon collection, stabilize samples in RNAprotect Tissue Reagent or AllProtect Tissue Reagent overnight. After overnight incubation, remove excess reagent before freezing. Snap freeze tissues in liquid nitrogen. Store samples frozen at -80 ºC, and ship on dry ice.

FFPE samples:

Consult QIAGEN Technical Services before isolating and submitting samples, and preferably before sample collection. For slide-mounted tissue sections, submit at least 4 slides with at least 5- to 10-micron thick sections. More slides may be requested from certain tissue sources, if available. For freshly cut sections of whole FFPE blocks, submit three to four 20-micron thick sections. Specify fixation procedure and age of samples. Store and ship at ambient temperature.

LCM samples:

Consult QIAGEN Technical Services before isolating and submitting samples. Store samples frozen at -80 ºC, and ship on dry ice.

FNAB and other small samples:
Consult QIAGEN Technical Services before isolating and submitting samples.

Other sample types:

Consult QIAGEN Technical Services before isolating and submitting samples.

Submitting purified RNA

RNA samples are very sensitive to RNase digestion. Wear gloves and maintain an RNase-free work area while purifying RNA.

Recommended RNA purification methods
Sample type Method
Cultured cells miRNeasy Kit or RNeasy Plus Kit (used with QIAGEN Supplementary Protocol: Purification of miRNA from animal cells using the RNeasy Plus Mini Kit and RNeasy MinElute Cleanup Kit) or RNeasy Plus Micro Kit
Tissue miRNeasy Kit
FNAB miRNeasy Kit
FFPE samples miRNeasy FFPE Kit
LCM samples miRNeasy FFPE Kit for samples from FFPE sections; miRNeasy Kit for samples from cryosections
Small samples yielding less than 100 ng total RNA miRNeasy Kit
Whole blood Before RNA preparation, red blood cells must be removed from whole blood samples using either selective lysis (e.g., as described in the QIAamp RNA Blood Mini Handbook) or a density gradient centrifugation medium (e.g., Lymphoprep, Greiner Bio-One [cat. no. 1031966]). RNA should then be purified from the white blood cell fraction using the miRNeasy Mini Kit. Alternative methods of RNA purification include use of the PAXgene Blood RNA Kit.
Total RNA isolated using a phenol-based method Clean up the RNA using an miRNeasy Kit
Other sample types Contact QIAGEN Technical Services
 

For best results from the PCR Array, all RNA samples should be suspended in the RNase-free water provided with the RNA Isolation kit. Do not use DEPC-treated water.

Note: Total RNA preps are highly suitable for use with the miScript miRNA PCR Array Service. It is not necessary to provide small RNA-enriched preps.

Quality control

We recommend verifying the quality of purified RNA using UV spectrophotometry. Please submit the results to the miScript miRNA PCR Array Service for evaluation.
To assess RNA quality by examining ribosomal RNA band integrity, electrophorese a fraction of each RNA sample on a denaturing agarose gel or on the QIAxcel.

Note: Due to the fragmentation associated with formalin fixation and paraffin embedding, RNA isolated from FFPE blocks and slides is always degraded, and the extent of degradation varies among samples. Therefore, while the integrity of RNA isolated from FFPE blocks or slides may be determined by the methods described here, it may only be useful for the purposes of comparing samples.

Note: RNA yields from some sources, such as serum or plasma samples, LCM samples, and FNAB samples are too low to enable quality control by these methods.

Data delivery

Data analysis is performed using QIAGEN data analysis software. Data in an electronic form can be downloaded from a secure FTP Website or delivered via email. A complete data and experiment report includes:

Raw CT valuesΔΔCT AnalysisExcel spreadsheet (data analysis using pair-wise comparisons, including raw data, tabular results as well as Scatter, 3D, and Volcano plots)

Applications

miScript miRNA PCR Arrays are used as part of the miScript PCR System for:

Pathway- and disease-focused miRNA profiling
miRNome profiling
Biomarker discovery

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miScript II RT Kit

miScript SYBR Green PCR Kit

miScript miRNA PCR Arrays

miScript miRNA QC PCR Array

For SYBR Green-based, real-time PCR profiling of microRNAs using pathway-focused arrays, HC arrays, and miRNome arrays


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仪器技术参数
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For pathway-focused miRNA or miRNome analysis
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For gene expression and genomic analysis
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下载文件
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Data analysis file for miScript miRNA PCR Array All miRNA QC (96-well plate, Rotor-Disc 100)
Catalog number- 331221
Pathway number- MIXX-989Z
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图片
Pathway-Focused or miRNome miScript miRNA PCR Array layout for plate formats A, C, D, F
按照信号通路或miRNome排列的miScript miRNA PCR Array布局。
A1至G12孔(1–84)分别含有一种针对通路/疾病/功能相关的成熟miRNA的miScript Primer Assay。H1和H2孔含有重复的C. elegans miR-39 miScript Primer Assay,也可用作芯片数据的标准化(Ce)。H3至H8孔分别含有一种针对不同snoRNA/snRNA的分析,可用作芯片数据的标准化对照(SN1=SNORD61 assay,SN2=SNORD68 assay,SN3=SNORD72 assay,SN4=SNORD95 assay,SN5=SNORD96A assay,SN6=RNU6B/RNU6-2 assay)。H9和H10孔含有重复的逆转录对照(miRTC)。H11和H12孔含有重复的阳性PCR对照(PPC)。
miRNome miScript miRNA PCR Array layout for plate formats E, G
miRNome miScript miRNA PCR Array和miScript miRNA HC PCR Array的孔板E、G布局
A1至P12孔(1–372)分别含有一种针对通路/疾病/功能相关的成熟miRNA的miScript Primer Assay。P13和P14孔含有重复的C. elegans miR-39 miScript Primer Assay,也可用作芯片数据的标准化(Ce)。P15至P20孔分别含有一种针对不同snoRNA/snRNA的分析,可用作芯片数据的标准化对照(SN1=SNORD61 assay,SN2=SNORD68 assay,SN3=SNORD72 assay,SN4=SNORD95 assay,SN5=SNORD96A assay,SN6=RNU6B/RNU6-2 assay)。P21和P22孔含有重复的miRTC miScript Primer Assay(miRTC)。P23和P24孔含有重复的阳性PCR对照(PPC)。
Pathway-Focused miScript miRNA PCR Array layout for plate formats E, G
Pathway-Focused miScript miRNA PCR Array的E、G规格孔板布局。
E和G形式的Pathway-Focused miScript miRNA PCR Array中包括与96孔A、C、D和F形式中相同的试剂,每种试剂重复4个。
miScript miRNA PCR Array layout for Rotor-Disc format R
miScript miRNA PCR Array的Rotor-Disc转子布局。
1至84孔分别含有一种针对通路/疾病/功能相关基因的miScript Primer Assay。85和86孔含有重复的C. elegans miR-39 miScript Primer Assay,也可用作芯片数据的标准化(Ce)。87至92孔分别含有一种针对不同snoRNA/snRNA的分析,可用作芯片数据的标准化对照(SN1=SNORD61 assay,SN2=SNORD68 assay,SN3=SNORD72 assay,SN4=SNORD95 assay,SN5=SNORD96A assay,SN6=RNU6B/RNU6-2 assay)。93和94孔含有重复的miRTC miScript Primer Assay(miRTC)。95和96孔含有重复的阳性PCR对照(PPC)。97–100孔为空。
miScript miRNA QC PCR Array layout for plate formats A, C, D, F.
miScript miRNA QC PCR Array的A、C、D、F规格孔板布局。
每排的1和2孔含有重复的C. elegans miR-39 miScript Primer Assay(39)。每排的3孔含有miR-16 miScript Primer Assay(16)。每排的4孔含有miR-21 miScript Primer Assay(21)。每排的5孔含有miR-191 miScript Primer Assay(191)。每排的6至8孔分别含有一种针对不同snoRNA的分析(61 = SNORD61 assay,95 = SNORD95 assay,96A = SNORD96A assay)。每排的9和10孔含有重复的miRTC miScript Primer Assay(miRTC)。每排的11和12孔含有重复的阳性PCR对照(PPC)。上述形式可实现至多8个cDNA样本的质量评估。
miScript miRNA QC PCR Array layout for plate formats E and G.
miScript miRNA QC PCR Array的E和G规格孔板布局。
每排的1至4孔含有重复的C. elegans miR-39 miScript Primer Assay(39)。每排的5和6孔含有重复的miR-16 miScript Primer Assay(16)。每排的7和8孔含有重复的miR-21 miScript Primer Assay(21)。每排的9和10孔含有重复的miR-191 miScript Primer Assay(191)。每排的11至16孔分别含有一种针对不同snoRNA的分析(61 = SNORD61 assay,95 = SNORD95 assay,96A = SNORD96A assay)。每排的17至20孔含有重复的miRTC miScript Primer Assay(miRTC)。每排的21至24孔含有重复的阳性PCR对照(PPC)。上述形式可实现至多32个cDNA样本的质量评估。
miScript miRNA QC PCR Array layout for Rotor-Disc format R.
miScript miRNA QC PCR Array的Rotor-Disc转子布局。
1和2孔含有重复的C. elegans miR-39 miScript Primer Assay。3孔含有miR-16 miScript Primer Assay。4孔含有miR-21 miScript Primer Assay。5孔含有miR-191 miScript Primer Assay。6至8孔分别含有一种针对不同snoRNA的分析(6孔 = SNORD61 assay,7孔 = SNORD95 assay,8孔 = SNORD96A assay)。9和10孔含有重复的miRTC miScript Primer Assay(miRTC)。11和12孔含有重复的阳性PCR对照(PPC)。13至96孔重复上述排列7次。97至100孔为空。上述形式可实现至多8个cDNA样本的质量评估。
Effortless data analysis
全miRNA组谱分析。
从采用± 5-氮杂-2'-脱氧胞苷(5-aza-2'-dC)去甲基试剂处理的HCT 116结直肠癌细胞中提取总RNA。该试剂通过掺入DNA并与DNA甲基转移酶的活性位点共价结合,不可逆地抑制DNA甲基转移酶介导的甲基化反应。使用miScript II RT Kit及miScript HiSpec Buffer制备cDNA。Human miRNome miScript miRNA PCR Array与miScript SYBR® Green PCR Kit结合使用,通过real-time PCR进行成熟miRNA的表达谱分析。使用在线miScript miRNA PCR Array Data Analysis Tool进行数据分析。[A] 2-ΔCT值散点图显示,2种样本中成熟miRNA的表达水平存在明显差异。[B] 火山图显示了调控miRNA(以±4倍为阈值,并以红线表示)。在5-aza-2'-dC处理的HCT 116细胞中,104种miRNA出现明显上调,30种明显下调。当p值为0.05时(以蓝线表示),104种miRNA中的89种上调明显,30种miRNA中的21种下调明显。
Analysis of expression of circulating miRNAs in serum or plasma holds huge potential as a source of new biomarkers
对血清或血浆中的游离miRNA表达进行分析,发现潜在的新生物标记物。
散点图显示了使用miScript miRNA PCR Array,健康血清样本和结肠癌血清样本的混合RNA中的相对miRNA表达水平。