Poly(A) Tail Length Measurement by PCR
First Claim
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1. A method for estimating the length of the polyA tail of a target mRNA comprising:
- adding a guanosine-inosine tail to the 3′
end of the target mRNA;
hybridizing a first primer to the guanosine-inosine tail and extending said first primer to obtain a primer extension product comprising a complement of the polyA tail of the target mRNA;
amplifying the primer extension product by PCR using a universal primer and a target specific primer to obtain a PCR product;
estimating the length of the PCR product; and
,estimating the length of the polyA tail of the target mRNA from the estimated length of the PCR product.
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Abstract
Methods and kits for measuring the length of the poly(A) tail of selected target mRNA are disclosed herein. In preferred aspects the mRNA population is modified by addition of a tail comprising guanosine and inosine (G/I tailing). The added tail is used as a priming site for reverse transcription. The resulting cDNA is then amplified using one or more target specific primers and a universal primer that recognizes the tailed region. The products are separated according to size and the size is used to estimate the polyA tail length.
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15 Claims
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1. A method for estimating the length of the polyA tail of a target mRNA comprising:
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adding a guanosine-inosine tail to the 3′
end of the target mRNA;hybridizing a first primer to the guanosine-inosine tail and extending said first primer to obtain a primer extension product comprising a complement of the polyA tail of the target mRNA; amplifying the primer extension product by PCR using a universal primer and a target specific primer to obtain a PCR product; estimating the length of the PCR product; and
,estimating the length of the polyA tail of the target mRNA from the estimated length of the PCR product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 14, 15)
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8. A kit comprising a reverse transcription primer that comprises between 2 and 5 contiguous T bases at the 3′
- end and between 8 and 15 contiguous C bases at the 3′
end. - View Dependent Claims (9, 10, 11, 12, 13)
- end and between 8 and 15 contiguous C bases at the 3′
Specification