Biomedical subjects

Keqin Gregg

Publications and source records attributed to Keqin Gregg.

4 recordsLinked to original sources

PubMed2004

A method for gene expression analysis by oligonucleotide arrays from minute biological materials.

Gene expression profiling has been widely used in identifying differentially expressed genes. One of the most popular formats is oligonucleotide array. A limitation of oligonucleotide arrays is the requirement of relatively large amounts of biological starting materials for gene expression analysis. We have developed a simple method for gene expression profiling from very small amounts of biological material by combining exponential (PCR) and linear (T7 RNA polymerase) amplification. By modifying the widely used SMART protocol, we combined T7 promoter ligation and PCR amplification in one step and generated around 0.5 microg of PCRcDNA from 30 ng of total RNA in a single PCR. The PCRcDNA was in vitro transcribed by T7 RNA polymerase to generate complementary RNA (cRNA), which then was used to hybridize Affymetrix GeneChips. Our results demonstrated a linear correlation between the PCR amplification and the conventional linear amplification in gene expression ratios of individual transcript species between two different RNA preparations. The method was further validated by TaqMan reactions. Moreover, both linear and PCR methods showed some inherent bias as to which transcripts were detected, suggesting that using both in parallel may provide a more comprehensive coverage of the transcriptome present in a given sample.

Base Sequence↗

PubMed2004

A method for cross-species gene expression analysis with high-density oligonucleotide arrays.

DNA microarrays have been widely used in gene expression analysis of biological processes. Due to a lack of sequence information, the applications have been largely restricted to humans and a few model organisms. Presented within this study are results of the cross-species hybridization with Affymetrix human high-density oligonucleotide arrays or GeneChip using distantly related mammalian species; cattle, pig and dog. Based on the unique feature of the Affymetrix GeneChip where every gene is represented by multiple probes, we hypothesized that sequence conservation within mammals is high enough to generate sufficient signals from some of the probes for expression analysis. We demonstrated that while overall hybridization signals are low for cross-species hybridization, a few probes of most genes still generated signals equivalent to the same-species hybridization. By masking the poorly hybridized probes electronically, the remaining probes provided reliable data for gene expression analysis. We developed an algorithm to select the reliable probes for analysis utilizing the match/mismatch feature of GeneChip. When comparing gene expression between two tissues using the selected probes, we found a linear correlation between the cross-species and same-species hybridization. In addition, we validated cross-species hybridization results by quantitative PCR using randomly selected genes. The method shown herein could be applied to both plant and animal research.

Algorithms↗

PubMed2004

Enhanced sensitivity RNA gel loading buffer that enables efficient RNA separation on native gels.

RNA gel analysis is essential for quality assessment of RNA preparations for subsequent analysis such as microarrays and real-time PCRs. The routinely used standard electrophoresis of RNA through formaldehyde-containing agarose gels is not only labor-intensive and time-consuming, but also involves sizeable quantities of hazardous materials. Above all, it is not sensitive, requiring more than 1 microgram of RNA for the assay. Current gene expression profiling with microarrays and real-time PCR often involves limiting amounts of RNA. It is therefore important to have a more sensitive way to analyze RNA. Here we report an improved ethidium bromide-based RNA gel analysis system with our Superload buffer that increases sensitivity to 12.5 ng of total RNA and allows RNA analysis on a regular native Tris-acetate EDTA (TAE) agarose gel.

Animals↗

PubMed2003

Binding of CCAAT displacement protein CDP to adenovirus packaging sequences.

Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis-acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding activities to Ad DNA packaging. The results of these experiments reveal that COUP-TF and Oct-1 binding does not play a functional role in Ad packaging, whereas P-complex binding directly correlates with packaging function. We demonstrate that P complex contains the cellular protein CCAAT displacement protein (CDP) and that full-length CDP is found in purified virus particles. In addition to cellular factors, previous evidence indicates that viral factors play a role in the initiation of viral DNA packaging. We propose that CDP, in conjunction with one or more viral proteins, binds to the packaging sequences of Ad to initiate the encapsidation process.

Adenoviruses, Human↗