Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-09-05
2001-02-06
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S091500, C435S091510, C435S006120, C436S094000, C436S503000, C536S023100, C536S024200, C536S024300, C536S024330, C536S025320
Reexamination Certificate
active
06183995
ABSTRACT:
BACKGROUND OF THE INVENTION
Wax-embedded tissue samples are convenient reservoirs of nucleic acid information. The presence of mRNA in such samples can provide extremely useful information about the expression of genes in certain cell types that have been preserved in this manner. However, when efforts have been made to obtain information about the mRNA in such samples, it is clear that extensive degradation of the cellular RNA exists.
Some researchers have reported detection of mRNA in such preserved tissues by in situ hybridization. Efforts have been made to extract mRNA from such samples and individual species of certain mRNA have been detected by PCR, however, these efforts are generally limited because the mRNA is degraded to small fragments usually less than 200 or 250 nucleotides in length. Because of the extent of degradation of the mRNA, efforts to attempt to obtain a representation of all species of mRNA from such samples have been deemed to be futile.
Further, about 95% of cellular RNA is ribosomal RNA (rRNA). Separation of the mRNA from the rRNA is difficult in samples such as these. The rRNA needs to be eliminated in gene expression studies because it does not reflect expression of the cellular genes. Common methods for separating mRNA from rRNA, such as the oligo (dT) method, operate by selecting for polyadenylated RNA. These methods are not very effective in these types of preserved cell samples. They automatically exclude much of the mRNA because the degradation which has occurred in the sample results in a high percentage of mRNA that does not have a polyA tail.
Also, when RNA is being studied, DNA must be removed to prevent background problems that result from its presence. Though RNA is typically separated from DNA by precipitating it away from the DNA, this method has the disadvantage that the RNA fragments which are obtained are of the same or similar size thus, limiting the representation of the total RNA population. Since paraffin-embedded tissues are a relatively convenient source of a variety of tissue types, it would be highly desirable to develop a method of obtaining a complete representation of the cellular mRNA from paraffin-embedded tissue samples for genomic analysis.
SUMMARY OF THE INVENTION
Our methods for extraction of mRNA from wax-embedded tissues and methods for production of resultant cDNA probes from such mRNA overcome the problems in the prior art, in part, by taking advantage of the fragmentation and degradation of the resident mRNA. The present degraded fragments of mRNA are readily copied and amplified without bias or omission. The methods described here can provide a complete representational library of cellular mRNA which can be copied to cDNA for multiple uses such as probes. The cDNA represents a reflection of the genes which are expressed in the source material and enables the genomic analysis of such material. Further, it was surprising that our methods did not result in much contamination from rRNA since none of the steps in the method are specifically directed to its removal, or at least not in the traditional way through the selection of polyadenylated RNA. Our methods also demonstrate that DNase may effectively be used in combination with our extraction method to reduce the background noise caused by DNA.
Specifically, the invention relates to a method for the production of cDNA representative of complete representational cellular mRNA from a wax-embedded biological sample wherein the RNA is extracted from the wax-embedded sample by removing the DNA in the biological sample with a DNAse and without a step to selectively eliminate rRNA by selection for polyadenylated RNA to produce sample cellular RNA. A strand of cDNA complementary to the sample cellular RNA is synthesized by contacting the sample RNA to one or more oligonucleotides and a reverse transcriptase to create single strand cDNA copies of the cellular RNA. The cDNA may then optionally be isolated by selectively separating the cellular RNA from the cDNA copies. The cDNA may be directly useful as probes, particularly if it is synthesized using labeled oligonucleotides or dNTPs. Complementary strands of the cDNA copies may be synthesized to create double-stranded cDNA. The double-stranded cDNA is preferably amplified by ligating a pair of complementary oligonucleotide adaptors to the double-stranded cDNA. The adaptors will be such that they are of different lengths. The cDNA copies may be amplified with a primer complementary to one of the adaptors.
The objectives of the methods of this invention are accomplished best when the adaptors used are blunt on one end and staggered on the other. The use of such adaptors results in an unbiased amplification of the cDNA and provides a more accurate picture of the total resident cellular mRNA. Each resident cDNA fragment is equally likely to have the blunt adaptors ligated to it and thus, to be amplified. The use of adaptors that are sticky on both ends may be useful in some cases, for example where the source mRNA was not very degraded, but is less preferred where total cellular mRNA is truly desirable.
Thus, the methods herein will imply many uses for the resulting mRNA or cDNA. The mRNA from the wax-embedded samples can be employed in comparison assays with mRNA or cDNA from other wax-embedded samples or with mRNA, cDNA or DNA from other samples of known or unknown origin depending upon the information desired. Methods for determining the presence or absence of gene expression in a wax-embedded biological sample are described comprising screening cDNA probes derived from mRNA extracted from wax-embedded biological material against nucleic acid material containing a known gene of interest. The cDNA may also be screened against cDNA from other wax-embedded samples or other nucleic acid source to determine if the same nucleic acid is expressed in both samples. All of such screening can be advantageously conducted in a high density array arrangement and may be conducted against nucleic acid representative of different tissue types.
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Brown Joseph P.
Burmer Glenna C.
Ford Amanda A.
Achutamurthy Ponnathapu
LifeSpan BioSciences, Inc.
Moore William W.
Townsend and Townsend / and Crew LLP
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