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miScript miRNA PCR Array Layouts


96-well Pathway-Focused or 96-well miRNome miScript miRNA PCR Array layout

Pathway-Focused or miRNome miScript miRNA PCR Array layout for plate formats A, C, D, F. 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 miRTC miScript Primer Assays (miRTC). Wells H11 and H12 contain replicate positive PCR controls (PPC).

 


Rotor-Disc 100 miScript miRNA PCR Array layout

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.


Pathway-Focused 384-well (4 x 96) miScript miRNA PCR Array layout

The 384-well plate format for the Pathway miScript miRNA PCR Arrays includes four replicates of the 96-well Pathway-Focused miScript miRNA PCR Array.

Pipetting PCR pre-mix into this array is straightforward and easy using the 384EZLoad Covers.



miScript miRNA HC PCR Array and 384-well miRNome miScript miRNA PCR Array layout

Wells A1 to P12 (1–372) each contain a miScript Primer Assay for a miRNome or 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).

Custom miScript miRNA PCR Arrays can be manufactured to almost any specified layout.

miScript  miRNA PCR Array System Controls

C. elegans miR-39 miScript Primer Assay

The miScript Primer Assay for C. elegans miR-39 detects the miRNeasy Serum/Plasma Spike-In Control (cat. no. 219610), (also called Syn-cel-miR-39 miScript miRNA Mimic [cat. no. MSY0000010]), which is a C. elegans miR-39 mimic. This mimic can be added to samples, particularly serum or plasma samples, to control for variations during the preparation of total RNA and subsequent steps. After purification, real-time RT-PCR detection of the miRNeasy Serum/Plasma Spike-In Control can be performed and these results can then be used for normalization of real-time RT-PCR results for endogenous miRNAs in the sample.

snoRNA/snRNA miScript PCR Controls

For accurate and reproducible results in miRNA quantification by real-time PCR, it is necessary to normalize the amount of target miRNA by using a suitable endogenous reference RNA. This approach is known as relative quantification. Normalization corrects for factors that could otherwise lead to inaccurate quantification. These factors include variation in quantity of input RNA, possible RNA degradation or presence of inhibitors in the RNA samples, and differences in sample handling. Normalization also allows results from different experiments and samples to be compared directly. miScript PCR Controls are primers designed to quantify a panel of 5 snoRNAs (SNORD61, SNORD68, SNORD72, SNORD95, and SNORD96A) and the snRNA RNU6B (RNU6-2). These controls take into consideration sequence homologies in human, mouse, rat, dog, and rhesus macaque so that the same controls can be used for all 4 species. In addition, these small RNAs have been verified to have relatively stable expression levels across tissues and cell types. As a result, miScript PCR Controls serve as normalization controls for relative quantification using the miScript PCR System. All the controls have amplification efficiencies close to 100%.

Reverse transcription control

The miRTC miScript Primer Assay is an assay that assesses the performance of a reverse-transcription reaction using the miScript II RT Kit by detecting template synthesized from the kit’s built-in miRNA reverse transcription control RNA (miRTC). This control monitors for any variables that may inhibit the reverse transcription reaction.

Positive PCR control

The positive PCR control (PPC) wells contain a predispensed artificial DNA sequence and the assay that detects it. This control monitors for any variables that may inhibit the PCR reaction.

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