TUCSON, Ariz.—High Throughput Genomics Inc. (HTG), announced last month it will use its quantitative Nuclease Protection Assay (qNPA) in a collaboration with the John Wayne Cancer Institute (JWCI) at Saint John's Health Center in Santa Monica, Calif., to generate a novel mRNA signature that can measure the gene expression differences in melanoma and benign nevi using classical surgical pathology samples.
"The program will provide new approaches to improve diagnosis of early-stage, primary cutaneous melanomas. This area of diagnosis has been problematic whereby novel sensitive molecular diagnosis approaches may be more informative and accurate," says Dr. Dave S. Hoon, director of JWCI's department of molecular oncology.
Hoon is investigating the implications of differential gene expression in human diseases such as melanoma. The ability to distinguish between early-stage primary melanomas and benign nevi is important given the difficulty that is sometimes encountered in diagnosing primary melanoma and, in turn, managing the disease better before onset of clinical metastasis.
"Our work with Dr. Hoon in this research program will further establish HTG's qNPA technology as a useful and robust validation and diagnostic platform. We believe our multiplex gene expression platform coupled with our extraction free method of analyzing formalin fixed paraffin embedded (FFPE) samples makes us ideally suited for this important application," said T.J. Johnson, president and CEO of HTG.
According to B.J. Kerns, senior vice president of operations at HTG, her company was making a routine sales call at JWCI when the question was raised about a procedure that would analyze for melanoma in FFPE samples, mimicking PCR and fresh tissue in order to understand the typical nevi environment and the transformative process to early melanoma. She notes that qNPA doesn't require extraction that degrades or breaks up RNA, making qNPA technology uniquely suited to produce robust gene expression results from FFPE and other fixed tissues where RNA quality is an issue. These samples typically yield poor gene expression results due to RNA cross-linking and fragmentation; at best, significant time and resources are required to prepare the samples. Damaged or fragmented RNA also causes small, incomplete cDNA transcripts to be formed. Most techniques will also suffer from a strong 3′ bias in the data analysis results. qNPA does not require cDNA synthesis, and the qNPA oligonucleotides can be arranged throughout the transcript. If the RNA fragments are over ~75 bases, qNPA is unaffected by RNA fragmentation.
In addition, much of the RNA in fixed tissue samples is bound to large pieces of cellular debris preventing it from being soluble and available for purification, causing large amounts of FFPE tissues to be wasted. qNPA does not use an RNA extraction prior to analysis. qNPA oligos can enter cellular debris complexes, be protected by non-soluble, cross-linked RNA from S1 nuclease digestion, and collected for measurement on the Array Plate platform. Less sample is needed and none is wasted. Finally, common contaminants such as paraffin, melanin, and polysaccharides are often carried over in RNA extraction techniques, inhibiting polymerases and causing poor results to be obtained. The qNPA process effectively removes any paraffin in a sample with an oil-overlay step. The technology does not use polymerases or other enzymes which may be inhibited by polysaccharides.
Privately held HTG was founded by Dr. Bruce E. Seligmann, who began his scientific career at the NIH-NIAID, Laboratory of Clinical Investigation, and then joined Ciba-Geigy (Novartis). In 1997, he formed HTG, at first serving pharma with front-end studies before venturing into qNPA's suitability for FFPE investigations that now comprise about 60 percent of the company's customer baser.
"The program will provide new approaches to improve diagnosis of early-stage, primary cutaneous melanomas. This area of diagnosis has been problematic whereby novel sensitive molecular diagnosis approaches may be more informative and accurate," says Dr. Dave S. Hoon, director of JWCI's department of molecular oncology.
Hoon is investigating the implications of differential gene expression in human diseases such as melanoma. The ability to distinguish between early-stage primary melanomas and benign nevi is important given the difficulty that is sometimes encountered in diagnosing primary melanoma and, in turn, managing the disease better before onset of clinical metastasis.
"Our work with Dr. Hoon in this research program will further establish HTG's qNPA technology as a useful and robust validation and diagnostic platform. We believe our multiplex gene expression platform coupled with our extraction free method of analyzing formalin fixed paraffin embedded (FFPE) samples makes us ideally suited for this important application," said T.J. Johnson, president and CEO of HTG.
According to B.J. Kerns, senior vice president of operations at HTG, her company was making a routine sales call at JWCI when the question was raised about a procedure that would analyze for melanoma in FFPE samples, mimicking PCR and fresh tissue in order to understand the typical nevi environment and the transformative process to early melanoma. She notes that qNPA doesn't require extraction that degrades or breaks up RNA, making qNPA technology uniquely suited to produce robust gene expression results from FFPE and other fixed tissues where RNA quality is an issue. These samples typically yield poor gene expression results due to RNA cross-linking and fragmentation; at best, significant time and resources are required to prepare the samples. Damaged or fragmented RNA also causes small, incomplete cDNA transcripts to be formed. Most techniques will also suffer from a strong 3′ bias in the data analysis results. qNPA does not require cDNA synthesis, and the qNPA oligonucleotides can be arranged throughout the transcript. If the RNA fragments are over ~75 bases, qNPA is unaffected by RNA fragmentation.
In addition, much of the RNA in fixed tissue samples is bound to large pieces of cellular debris preventing it from being soluble and available for purification, causing large amounts of FFPE tissues to be wasted. qNPA does not use an RNA extraction prior to analysis. qNPA oligos can enter cellular debris complexes, be protected by non-soluble, cross-linked RNA from S1 nuclease digestion, and collected for measurement on the Array Plate platform. Less sample is needed and none is wasted. Finally, common contaminants such as paraffin, melanin, and polysaccharides are often carried over in RNA extraction techniques, inhibiting polymerases and causing poor results to be obtained. The qNPA process effectively removes any paraffin in a sample with an oil-overlay step. The technology does not use polymerases or other enzymes which may be inhibited by polysaccharides.
Privately held HTG was founded by Dr. Bruce E. Seligmann, who began his scientific career at the NIH-NIAID, Laboratory of Clinical Investigation, and then joined Ciba-Geigy (Novartis). In 1997, he formed HTG, at first serving pharma with front-end studies before venturing into qNPA's suitability for FFPE investigations that now comprise about 60 percent of the company's customer baser.
