Quantification of mRNA in Salmonella sp. seeded soil and chicken manure using magnetic capture hybridization RT-PCR

Direct quantification of mRNA from Salmonella sp. seeded for 1 h to soil and chicken manure was accomplished using magnetic capture hybridization as a purification technique. This detection strategy targeted the invA gene present in Salmonella sp. After cell lysis, phenol/chloroform purification and isopropanol precipitation, the RNA extract was combined with the hybridization probe conjugated to paramagnetic beads. After hybridization, the captured nucleic acids were released by denaturation and purified of contaminating DNA using DNase. The resulting RNA was of high purity and there was no need for dilution of the samples prior to RT-PCR. The developed procedure was reproducibly used to quantify Salmonella sp. in high organic agricultural soil. The detection limit for mRNA using ordinary quantitative PCR (employing SYBRgreen-based detection) was 5 x 10(4)Salmonella sp. cells per gram of soil. Chicken manure amended into soil (1:4 w/w) did not reduce the ability to quantify Salmonella sp. mRNA in soil. Pasteurization (65 degrees C, 30 min) of chicken manure containing Salmonella sp. dramatically reduced the detection of invA mRNA (requiring 42 qPCR cycles for detection versus 26 cycles in unpasteurized manure), presumably due to degradation of the invA mRNA in Salmonella sp. cells killed by pasteurization. By contrast, DNA-based qPCR still detected Salmonella sp. in the pasteurized manure. Thus, in this case using samples seeded with fresh Salmonella sp. the mRNA-based detection appears to be superior to minimizing false-positive detection which was prevalent with DNA-based qPCR.     

RNA isolation and fractionation with compaction agents

A new approach to the isolation of RNA from bacterial lysates employs selective precipitation by compaction agents, such as hexammine cobalt and spermidine. Using 3.5 mM hexammine cobalt, total RNA can be selectively precipitated from a cell lysate. At a concentration of 2 mM hexammine cobalt, rRNA can be fractionated from low molecular weight RNA. The resulting RNA mixture is readily resolved to pure 5S and mixed 16S/23S rRNA by nondenaturing anion-exchange chromatography. Using a second stage of precipitation at 8 mM hexammine cobalt, the low molecular weight RNA fraction can be isolated by precipitation. Compaction precipitation was also applied to the purification of an artificial stable RNA derived from Escherichia coli 5S rRNA and to the isolation of an Escherichia coli-expressed ribozyme.     

A bacteriophage lambda DNA purification procedure suitable for the analysis of DNA from either large or multiple small lysates

A method for the efficient preparation of high quality bacteriophage lambda DNA from cleared lysates is described. Advantages of the method include high DNA yields (typically around 0.8 micrograms of DNA/1 ml of cleared lysate), speed of processing (approximately 2 h from lysate to DNA), economy, and the absence of any requirement for phenol or chloroform extractions. The technique involves the concentration of phage particles by standard polyethylene glycol precipitation followed by enzymatic treatment to remove contaminating RNA and DNA. Phage particles are then lysed with sodium dodecyl sulfate (SDS) at elevated pH and temperature. Contaminating protein/SDS complexes are rendered insoluble by the addition of potassium acetate and removed by centrifugation. The quality of the resultant DNA is comparable to that prepared by cesium chloride banding for all standard molecular biological purposes providing that spermidine is included in all restriction endonucleases digestions.     

An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification

Although PCR and RT-PCR provided a valuable approach for detection of pathogens, the high level of sensitivity of these assays also makes them prone to false positive results. In addition to cross-contamination with true positive samples, false positive results are also possible due to "carry-over" contamination of samples with amplicon DNA generated by previous reactions. To reduce this source of false positives, amplicon generated by reactions in which dUTP was substituted for dTTP can be degraded by uracil DNA glycosylase (UNG). UNG does not degrade RNA but will cleave contaminating uracil-containing DNA while leaving thymine-containing DNA intact. The availability of heat-labile UNG makes use of this approach feasible for RT-PCR. In this study, real-time RT-PCR was used to quantify UNG degradation of amplicon DNA and the effect of UNG on RNA detection. Using the manufacturers' recommended conditions, complete degradation of DNA was not observed for samples containing 250 copies of amplicon DNA. Doubling the UNG concentration resulted in degradation of the two lowest concentrations of DNA tested, but also resulted in an increase of 1.94 cycles in the C_T for RNA detection. To improve DNA degradation while minimizing the effect on RNA detection, a series of time, temperature and enzyme concentrations were evaluated. Optimal conditions were found to be 0.25 U UNG per 25 μl reaction with a 20 min, 30°C incubation prior to RT-PCR. Under these conditions, high concentrations of amplicon DNA could be degraded while the C_T for RNA detection was increased by 1.2 cycles.     

Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro.

In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.     

Recovery of Small DNA Fragments from Serum Using Compaction Precipitation

Background

While most nucleic acids are intracellular, trace amounts of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), including micro RNAs, can also be found in peripheral blood. Many studies have suggested the potential utility of these circulating nucleic acids in prenatal diagnosis, early cancer detection, and the diagnosis of infectious diseases. However, DNA circulating in blood is usually present at very low concentrations (ng/ml), and is in the form of relatively small fragments (<1,000 bp), making its isolation challenging.

Methods

Here we report an improved method for the isolation of small DNA fragments from serum using selective precipitation by quaternary ammonium compaction agents. A 151 bp fragment of double-stranded DNA from the Escherichia coli bacteriophage lambda served as the model DNA in our experiments. DNA was serially diluted in serum until undetectable by conventional polymerase chain reaction (PCR), before being enriched by compaction precipitation.

Results

Starting with concentrations two to three orders of magnitude lower than the PCR-detectable level (0.01 ng/ml), we were able to enrich the DNA to a detectable level using a novel compaction precipitation protocol. The isolated DNA product after compaction precipitation was largely free of serum contaminants and was suitable for downstream applications.

Conclusions

Using compaction precipitation, we were able to isolate and concentrate small DNA from serum, and increase the sensitivity of detection by more than four orders of magnitude. We were able to recover and detect very low levels (0.01 ng/ml) of a small DNA fragment in serum. In addition to being very sensitive, the method is fast, simple, inexpensive, and avoids the use of toxic chemicals.     

Rapid and Reliable Method of Extracting DNA and RNA from Sweetpotato, Ipomoea batatas (L). Lam

A quick, simple and reliable method of extracting DNA from sweetpotato (Ipomoea batatas (L.) Lam.) has been developed. The method was applied successfully for extraction of total DNA from leaves and total RNA from leaves and various tissues. The yield of DNA extracted by this procedure was high (about 1 mg/g leaf tissue). The extracted DNA was completely digested by restriction endonucleases indicating the absence of common contaminating compounds. The absorbancy ratios of A260/A230 and A260/A280 of isolated RNA were approx. 2 and the yield was about 0.2 mg/g fresh wt. CIPK and tublin genes were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total DNA and RNA isolated by this method was of sufficient quality for subsequent molecular analysis.     

Inhibition of lymphocyte growth by spermidine in medium containing fetal bovine serum

The effect of spermidine and fetal bovine serum on DNA, RNA, and protein synthesis in phytohemagglutinin-stimulated human lymphocytes was investigated. At 10(-4) M spermidine, DNA, RNA, and protein synthesis ceased and 70% of the original cell population died within 62 hr. Lower spermidine concentrations had no significant effect on DNA and protein synthesis, but caused an early, unexplained increase in the rate of RNA synthesis. Heating at 56 degrees C for 30 min had no effect on the plasma amine oxidase activity in fetal bovine and horse sera but abolished the activity in human plasma. It is concluded that low amounts of aminoaldehydes and acrolein produced by plasma amine oxidase at spermidine concentrations below 10(-4) M do not noticeably alter lymphocyte metabolism. However, the aminoaldehydes and acrolein produced become abruptly cytotoxic at 10(-4) M spermidine.     

Bacterial DNA decontamination for reverse transcription polymerase chain reaction (RT-PCR)

For RT-PCR, removal of contaminating genomic DNA in RNA samples using manganese sulfate was more effective than magnesium. DNA contamination was removed in 3 microg of nucleic acid using 10 U of RNase-free DNase I in 10-microl reaction volumes. The digestion procedure was compatible with commercial RNA extraction kits and was suitable for RT-PCR assay.     

Poly(A) cDNA-specific (PACS) RT-PCR: a quantitative method for the measurement of any poly(A)-containing mRNA not affected by contaminating genomic DNA

We present a simple and efficient RT-PCR method for the detection and quantitation of any poly(A)-containing mRNA that is not affected by contaminating genomic DNA and does not rely on exhaustive DNase digestion protocols. The technique described here requires the use of an antisense primer designed to contain 6-8 bp cDNA-specific sequence and an additional 17 Ts located on the 5' end to take advantage of the poly(A) tail. A second cDNA-specific sense primer can be used that does not need to be separated by intronic DNA sequence.     

Monitoring of RNA clearance in a novel plasmid DNA purification process

As the field of plasmid DNA-based vaccines and therapeutics matures, improved methods for impurity clearance monitoring are increasingly valuable for process development and scale-up. Residual host-cell RNA is a major impurity in current large-scale separation processes for the production of clinical-grade plasmid DNA. Current RNA detection technologies include quantitative rtPCR, HPLC, and fluorescent dye-based assays. However, these methodologies are difficult to employ as in-process tests primarily as a result of impurity and buffer interferences. To address the need for a method of measuring RNA levels in various process intermediates, a sample pretreatment strategy has been developed that utilizes spermidine affinity precipitation to eliminate a majority of solution impurities, followed by a quantitative precipitation with alcohol to concentrate RNA and allow detection at lower concentrations. RNA concentrations as low as 80 ng/mL have been measured using detection with gel electrophoresis and 20 ng/mL if microplate-based detection with Ribogreen fluorescent dye is used. The assay procedure has been utilized to troubleshoot RNA clearance issues encountered during scale-up of a novel, non-chromatographic purification process for plasmid DNA. Assay results identified residual liquor removal inadequacies as the source of elevated RNA levels, enabling process modifications in a timely fashion.     

Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods

The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C. Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C. In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.