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Layout of the Cataloged 96-Well miRNA PCR Arrays
Wells A1 through H4 each contain one universal primer and one gene-specific
primer for 88 miRNA sequences related to the same biological pathway or the same
disease state, or the 88 most abundantly expressed and best characterized miRNA
sequences in the human genome.
Wells H5 through H8 contain a small RNA housekeeping gene panel to normalize
qPCR Array data including U6 and SNORD 44, 47, and 48.
The product specification sheet included with each cataloged qPCR Array
contains a list of the pathway-focused and housekeeping genes on the array.
Wells H9 and H10 contain duplicate Reverse Transcription Controls (RTC) to
test the efficiency of the miRNA Reverse Transcription Kit (MA-03) reaction with
a primer set detecting the template synthesized from the kit's built-in miRNA
External RNA Control (ERC).
Wells H11 and H12 contain duplicate Positive PCR Controls (PPC) to test the
efficiency of the polymerase chain reaction itself using a pre-dispensed
artificial DNA sequence and the primer set that detects it.
The two sets of duplicate control wells (RTC and PPC) also test for
inter-well, intra-plate consistency.
The 384-well format of the PCR Arrays includes four replicates of the same
96-well format, in which each two-by-two set of wells (wells labeled 1 - 4 in
gray above) contains the same primer set represented by the 96-well
designations.
Custom miRNA qPCR Arrays have your specified layout, and the product
information enclosed with the array reiterates your layout and the genes
included.
Layout of the Cataloged 384-Well miRNA PCR Arrays
Wells A1 through P16 each contain one universal primer and one gene-specific
primer for the 376 most abundantly expressed and best characterized miRNA
sequences in the human genome.
Wells P17 through P20 contain a small RNA housekeeping gene panel to
normalize qPCR Array data including U6 and SNORD 44, 47, and 48.
The product specification sheet included with each cataloged qPCR Array
contains a list of the pathway-focused and housekeeping genes on the array.
Wells P21 and P22 contain duplicate Reverse Transcription Controls (RTC) to
test the efficiency of the miRNA Reverse Transcription Kit (MA-03) reaction with
a primer set detecting the template synthesized from the kit's built-in miRNA
External RNA Control (ERC).
Wells P23 and P24 contain duplicate Positive PCR Controls (PPC) to test the
efficiency of the polymerase chain reaction itself using a pre-dispensed
artificial DNA sequence and the primer set that detects it.
The two sets of duplicate control wells (RTC and PPC) also test for
inter-well, intra-plate consistency.
Custom miRNA qPCR Arrays have your specified layout, and the product
information enclosed with the array reiterates your layout and the genes
included.
RT² miRNA PCR Array System Controls
The RT² miRNA PCR Arrays include built-in positive control elements
for the proper normalization of the data, for the quality of your RNA samples,
and for general PCR performance in your hands.
Housekeeping Genes:
Four wells contain a panel of assays for housekeeping small nuclear
RNA listed in the product specification sheet included with each cataloged or
custom PCR Array. Use the raw threshold cycle data from these assays to choose
an appropriate factor to normalize your PCR Array data, either the Ct value of
one housekeeping assay or the average value of up to all four of them. The
normalization factor should ideally not differ by more than one (1) cycle across
the compared samples.
Reverse Transcription Controls:
Two wells contain replicate Reverse Transcription Controls (RTC) to test
the efficiency of the RT² miRNA First Strand Kit (MA-03) reaction. The assay
detects template synthesized from the kit's built-in miRNA External RNA Control
(ERC). Any impurities that affect the reverse transcription of the ERC also
affect the reverse transcription of your experimental miRNA sample.
A value of (Ct RTC - Ct PPC) less than five (5) indicates no apparent
inhibition.
A value greater than five (5) provides evidence of impurities that inhibited the
reverse transcription phase of the procedure. Double check the quality, purity,
and integrity of the miRNA samples.
Positive PCR Controls:
Two wells contain replicate Positive PCR Controls (PPC) to test the
efficiency of the polymerase chain reaction itself. The assay includes a
pre-dispensed artificial DNA sequence and the primer set that detects it. Any
impurities that affect the positive control PCR amplification also affect
amplification of the gene-specific products of interest.
Values of Ct PPC = 20 +2 indicate a lack of PCR inhibitors.
Higher values and more widely variable values across arrays indicate the
presence of inhibitors with different concentrations in the
samples. Double check the quality, purity, and integrity of the RNA samples.
Also, double check that the correct cycling conditions recommended in the User
Manual were used.
The two sets of replicate control wells (RTC and PPC) on each RT² miRNA PCR
Array also test for inter-well, intra-plate consistency.
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