Extraction Of High-quality RNA Research Articles

Influence of trypsinization and alternative procedures for cell preparation before RNA extraction on RNA integrity

The accuracy of techniques such as microarrays, reverse transcription polymerase chain reaction, and whole transcriptome shotgun sequencing is critically dependent on RNA quality. We have repeatedly observed extensive RNA degradation following trypsinization, a routine procedure used to dissociate adherent tissue culture cells prior to RNA extraction. This study investigated the cause of this degradation and identifies an alternative procedure that enables extraction of intact high-quality RNA. Trypsinization and several alternative procedures were used to dissociate a range of different cell lines prior to RNA extraction. The contribution of exogenous ribonucleases or induction of endogenous ribonucleases by trypsin reagent proteases to RNA degradation was examined. Trypsinization resulted in a complete degradation of RNA regardless of cell line type, differentiation stage, or passage number. This occurred when intact RNA was incubated directly with trypsin and was not suppressed by inhibiting trypsin’s protease activity. Prevention of degradation by sodium hypochlorite treatment of trypsin reagent identified the presence of ribonucleases in trypsin derived from animal pancreas. Consistent extraction of high-quality RNA requires the use of direct cell lysis with a phenol guanidine-based reagent or an animal origin-free protease-based dissociation agent if enzymatic detachment prior to RNA extraction cannot be avoided.

Research note: methodology for high-quality RNA extraction from poultry whole blood for further gene expression analysis

1. There are no published methods for RNA isolation from avian whole blood where nucleated red blood cells prevent the use of established mammalian protocols. The aim of this study was therefore to develop a protocol for total RNA extraction using avian whole blood by defining the effect of anticoagulants and sample purification protocols on RNA yield and quality.2. Blood collections from the cutaneous ulnar or medial metatarsal veins of birds yielded adequate blood volume (2–3 ml) draws. The experiment was a 2 × 2 × 3 factorial arrangement of treatments, with two levels of DNase (0 and TURBO DNA-free Kit), two levels of Cleanup (0 and RNeasy MinElute Cleanup Kit), and three anticoagulants (no anticoagulant, EDTA, or sodium citrate).3. RNA was isolated successfully by adding TRIzol LS to 0.25 ml of chicken whole blood at 3:1 ratio. From 0.125 ml of avian whole blood, 2–3 µg of RNA with RNA integrity number values of 7.75 was successfully isolated with the TRIzol LS extraction and an RNeasy MinElute Cleanup Kit.4. This reliable protocol can be used to extract high yield and quality of total RNA from a small amount of whole blood.

Comparison of three different kits for extraction of high-quality RNA from frozen blood

Extraction of high-quality RNA is a crucial step in gene expression profiling. To achieve optimal RNA extraction from frozen blood, the performance of three RNA extraction kits- TRI reagent, PAXgene blood RNA system (PAXgene) and NucleoSpin RNA blood kit (NucleoSpin)- was evaluated. Fifteen blood specimens collected in tubes containing potassium ethylenediaminetetraacetic acid (EDTA) and stored at −80°C for approximately 5 years were randomly selected. The yield and purity of RNA, RIN (RNA integrity number) values and cycle threshold (Ct) values were assessed. Mean RNA yields with TRI reagent, PAXgene and NucleoSpin were 15.6 ± 8.7 μg/ml, 3.1 ± 1.7 μg/ml and 9.0 ± 5.5 μg/ml, respectively. Mean A260/280 ratios of RNA for the three kits were 1.7 ± 0.1, 2.0 ± 0.1, and 2.0 ± 0.0, and mean RIN values recorded as 3.2 ± 0.8, 6.0 ± 1.1, and 6.4 ± 0.9, respectively. The Ct values of housekeeping genes, 18S rRNA, β-actin, RPLP0 and HPRT1, were as follows: TRI reagent (19.2 ± 1.6, 30.6 ± 1.8, 29.9 ± 1.4 and 36.3 ± 1.3), PAXgene 16.6 ± 1.4, 26.4 ± 1.3, 28.2 ± 1.8 and 33.8 ± 1.1), and NucleoSpin (16.3 ± 1.5, 27.2 ± 1.3, 27.0 ± 1.6 and 32.9 ± 1.6). RNA yield using TRI reagent was 1.7 times higher than that with NucleoSpin and 5 times higher than that with PAXgene. However, the purity and integrity of TRI-extracted RNA was lower than that extracted with PAXgene and NucleoSpin. Moreover, the Ct values of housekeeping genes after extraction with TRI reagent were approximately 1.7-3.8 times higher than those obtained with PAXgene and NucleoSpin. The PAXgene and NucleoSpin kits produced similar results in terms of RNA purity and integrity and subsequent gene amplification. However, RNA yields from NucleoSpin were 2.9-fold higher, compared to PAXgene. Based on these findings, we conclude that NucleoSpin is the most effective kit for extraction of abundant and high-quality RNA from frozen blood.

Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani

800x600 Normal 0 21 false false false ES-CL X-NONE X-NONE Background: Rhi zoctonia solani (teleomorph: Thanatephorus cucumeris ) is one of the most important pathogens of rice ( Oryza sativa L.) that causes severe yield losses in all rice-growing regions. Sclerotia, formed from the aggregation of hyphae, are important structures in the life cycles of R. solani and contain a large quantity of polysaccharides, lipids, proteins and pigments. In order to extract high-quality total RNA from the sclerotia of R. solani , five methods, including E.Z.N.A. TM Fungal RNA Kit, sodium dodecyl sulfate (SDS)-sodium borate, SDS- polyvinylpyrrolidone (PVP), guanidinium thiocyanate (GTC) and modified Trizol, were compared in this study. Results: The electrophoresis results showed that it failed to extract total RNA from the sclerotia using modified Trizol method, whereas it could extract total RNA from the sclerotia using other four methods. Further experiments confirmed that the total RNA extracted using SDS-sodium borate, SDS-PVP and E.Z.N.A. TM Fungal RNA Kit methods could be used for RT-PCR of the specific amplification of GAPDH gene fragments, and that extracted using GTC method did not fulfill the requirement for above-mentioned RT-PCR experiment. Conclusion : It is concluded that SDS-sodium borate and SDS-PVP methods were the better ones for the extraction of high-quality total RNA that could be used for future gene cloning and expression studies, whereas E.Z.N.A. TM Fungal RNA Kit was not taken into consideration when deal with a large quantity of samples because it is expensive and relatively low yield. © 2014 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V. All rights reserved

Rapid and Reliable Method of High-Quality RNA Extraction from Diverse Plants

The isolation of high quality RNA is a crucial technique in plant molecular biology. The quality of RNA determines the reliability of downstream process like real time PCR. In this paper, we reported a high quality RNA extraction protocol for a variety of plant species. Our protocol is time effective than traditional RNA extraction methods. The method takes only an hour to complete the procedure. Spectral measurement and electrophoresis were used to demonstrate RNA quality and quantity. The extracted RNA was further used for cDNA synthesis, expression analysis and copy number determination through Real Time PCR. The results indicate that RNA was of good quality and fit for real time PCR. This high throughput plant RNA extraction protocol can be used to isolate high quality RNA from diverse plants for real time PCR and other downstream applications.

Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit crops

Strawberry plant tissues and particularly fruit material are rich in polysaccharides and polyphenolic compounds, thus rendering the isolation of nucleic acids a difficult task. This work describes the successful modification of a total RNA extraction protocol, which enables the isolation of high quantity and quality of total RNA from small amounts of strawberry leaf, root and fruit tissues. Reverse-transcription polymerase chain reaction (RT-PCR) amplification of GAPDH housekeeping gene from isolated RNA further supports the proposed protocol efficiency and its use for downstream molecular applications. This novel procedure was also successfully followed using other fruit tissues, such as olive and kiwifruit. In addition, optional treatment with RNase A following initial nucleic acid extraction can provide sufficient quality and quality of genomic DNA for subsequent PCR analyses, as evidenced from PCR amplification of housekeeping genes using extracted genomic DNA as template. Overall, this optimized protocol allows easy, rapid and economic isolation of high quality RNA from small amounts of an important fruit crop, such as strawberry, with extended applicability to other recalcitrant fruit crops.

A method for extracting high-quality RNA from diverse plants for next-generation sequencing and gene expression analyses.

• Premise of the study: To study gene expression in plants, high-quality RNA must be extracted in quantities sufficient for subsequent cDNA library construction. Field-based collections are often limited in quantity and quality of tissue and are typically preserved in RNAlater. Obtaining sufficient and high-quality yield from variously preserved samples is essential to studies of comparative biology. We present a protocol for the extraction of high-quality RNA from even the most recalcitrant plant tissues.• Methods and Results: Tissues from mosses, cycads, and angiosperm floral organs and leaves were preserved in RNAlater or frozen fresh at −80°C. Extractions were performed and quality was measured for yield and purity.• Conclusions: This protocol results in the extraction of high-quality RNA from a variety of plant tissues representing vascular and nonvascular plants. RNA was used for cDNA synthesis to generate libraries for next-generation sequencing and for expression studies using quantitative PCR (qPCR) and semiquantitative reverse transcription PCR (RT-PCR).

A SIMPLE METHOD FOR DETECTION OF POLYMYXA BETAE AND BEET NECROTIC YELLOW VEIN VIRUS IN SOIL

Extraction of high quality DNA and RNA from soil is a most challenging step to identify soil organisms due to the presence of a variety of inhibitors and microbial nucleases. In this study, we have developed a simple extraction method for simultaneous purification of DNA and RNA from soil. The isolated nucleic acids were of high quality allowing for sensitive detection of the oomycete Polymyxa betae and Beet necrotic yellow vein virus (BNYVV) by nested-PCR. The vector and BNYVV were detected in all infested soils but only the vector was detected in some apparently non-infested soils or fallow fields. The method may help in the management of sugar beet rhizomania.

High‐quality RNA extraction from rat pancreatic islet

In recent years, increasing interest surrounding islet replacement therapies in human has provided the drive for advances in the methods used to isolate from humans as well as a host of animal research models. However, there has been no reports describing a technique that reliably improves the quality of RNA extracted from rat pancreatic islet. Male Sprague–Dawley rats, 10- to 12-week-old, were housed in a certified animal care facility. The rats were underwent bile duct cannulation with pancreatic inflation after clamping the distal common bile duct. The pancreas was excised and digested with ETK/Liberase TL solution at 37°C for 30 min without shaking. Cold ETK was added to stop digestion. The islets were purified by discontinuous iodixanol density gradients of 25, 23, 20 and 11% in a modified ETK/OptiPrep® solution. After a 15 min centrifugation at 1,000g, islets were collected from the interface between the 20 and 11% layer. Immediately after purification, islets were used for RNA extraction. RNA was extracted from isolated rat pancreatic islet cells, using the commercially available kit. In the present study, we have described a technique that reliably improves the quality of RNA extracted from rat pancreatic islet using the perfusion technique in the bile duct. The islet cells that we isolated using this technique were suitable for high quality RNA extraction.

Extractions of High Quality RNA from the Seeds of Jerusalem Artichoke and Other Plant Species with High Levels of Starch and Lipid.

Jerusalem artichoke (Helianthus tuberosus L.) is an important tuber crop. However, Jerusalem artichoke seeds contain high levels of starch and lipid, making the extraction of high-quality RNA extremely difficult and the gene expression analysis challenging. This study was aimed to improve existing methods for extracting total RNA from Jerusalem artichoke dry seeds and to assess the applicability of the improved method in other plant species. Five RNA extraction methods were evaluated on Jerusalem artichoke seeds and two were modified. One modified method with the significant improvement was applied to assay seeds of diverse Jerusalem artichoke accessions, sunflower, rice, maize, peanut and marigold. The effectiveness of the improved method to extract total RNA from seeds was assessed using qPCR analysis of four selected genes. The improved method of Ma and Yang (2011) yielded a maximum RNA solubility and removed most interfering substances. The improved protocol generated 29 to 41 µg RNA/30 mg fresh weight. An A260/A280 ratio of 1.79 to 2.22 showed their RNA purity. Extracted RNA was effective for downstream applications such as first-stranded cDNA synthesis, cDNA cloning and qPCR. The improved method was also effective to extract total RNA from seeds of sunflower, rice, maize and peanut that are rich in polyphenols, lipids and polysaccharides.

TWO MODIFIED RNA EXTRACTION METHODS COMPATIBLE WITH TRANSCRIPT PROFILING AND GENE EXPRESSION ANALYSIS FOR COTTON ROOTS

Efficient isolation of high-quality RNA is of prime importance for optimal transcript profiling results and further gene expression analysis. However, it is difficult for cotton roots because of lower-than-average RNA content and high content of polysaccharides, polyphenols, and other secondary metabolites. To develop simple and reliable protocols for high-quality RNA extraction from cotton roots for transcript profiling and gene expression analysis, some modifications were introduced to a reported plant RNA isolation protocol and a reagent kit method. Using method A, we successfully extracted high-quality RNA for transcript profiling from cotton roots. Gel electrophoresis analysis and polymerase chain reaction (PCR) assay indicated that RNA had good integrity without protein and genomic DNA contamination. Furthermore, the A260/280 (1.9) and A260/230 (1.6) ratios indicated that the isolated RNA was of high purity. Using method B, about 7 µg total RNA of high quality could be obtained from 0.1 g samples from cotton roots, which can be used for reverse-transcription (RT)-PCR and quantitative real-time RT-PCR. The two RNA extraction methods were used to investigate different gene expression of cotton roots (Gossypium hirsutum) infected by weak pathogenic Verticillium dahliae and the results showed they can satisfy the transcript profiling and quantitative real-time RT-PCR requirements for RNA. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

Extraction of High-Quality RNA from Rubber Tree Leaves

A specific technique capable of producing high-quality RNA for rapid amplification of cDNA ends (RACE) was established for challenging tissues: leaves of the rubber tree. Total RNA was extracted by cetyltrimethylammonium bromide (CTAB)-LiCl combined with TRIzol reagent. The isolated RNA was highly intact. With RNA as template, full-length cDNA was obtained (NCBI, AY461413) by RACE.

A Simple and Swift Method for Isolating High Quality RNA from Jute (Corchorus spp.)

High quality RNA extraction is a prerequisite for various types of genetic analyses. Many a time, the existing RNA isolation methods and commercial kits are either time consuming or fail to isolate high quality RNA from plants rich in polysaccharides, oil and other secondary metabolites since different plants contain different amounts of nucleic acids (Khan et al. 2004, Loomis 1974). This problem is particularly acute in case of jute (Corchorus spp.), which is rich in mucilage and other polysaccharides that tends to interfere with the downstream processes (Kundu et al. 2011, Pandey et al. 1996). Several guanidium salt based methods have been successful for RNA isolation from jute seedlings (Khan et al. 2004), but are often cumbersome and expensive; hence limit simultaneous processing of large number of samples. Here we report a simplified and swift protocol for isolating high quality RNA from jute by making key modifications in tissue denaturation and precipitation steps in the protocol described by Ghawana et al. (2011). The protocol allows consistent production of high quality RNA from different species, which makes it particularly suitable for comparative plant genome research. The extraction time has been reduced from two days (for standard guanidium-acid-phenol extraction protocols) to about one hour and the extracted RNA was suitable for downstream processes like cDNA synthesis and expression pattern analysis. Key words: Jute, Corchorus spp., Swift method, RNA isolation, RT-PCR D. O. I. 10.3329/ptcb.v21i2.10244 Plant Tissue Cult. & Biotech. 21(2): 207-211, 2011 (December) - Short communication

English

RNA extraction is the foundation of modern molecular biology. It is difficult to extract high quality RNA from grapevine tissues, especially in cases where polysaccharides and polyphenols are rich. In this paper, the modified cetyltrie thylammnonium bromide (CTAB), sodium dodecyl sulphate (SDS) and guanidine isothiocyanate methods were applied to extract high purity and integrity RNA from grapevine green and mature berry skins. Highly-grade RNA was isolated by using modified CTAB method and it is suitable for the demands of further molecular biological research such as reverse-transcription polymerase chain reaction (PCR) (RT-PCR), cDNA library construction and Northern hybridization. Key words: Grapevine berry skins, RNA extraction, reverse-transcription polymerase chain reaction (RT-PCR).

An efficient RNA isolation procedure and identification of reference genes for normalization of gene expression in blueberry

Application of transcriptomics approaches can greatly enhance our understanding of blueberry physiology. The success of transcriptomics approaches is dependent on the extraction of high-quality RNA which is complicated by the abundance of polyphenolics and polysaccharides in blueberry. Additionally, transcriptomics requires the accurate quantification of transcript abundance. Quantitative real-time polymerase chain reaction (qRT-PCR) is a robust method to determine transcript abundance. Normalization of gene expression using stably expressed reference genes is essential in qRT-PCR. An evaluation of the stability of expression of reference genes has not yet been reported in blueberry. The objectives of this study were to develop an effective procedure for extracting RNA from different organs and to evaluate potential reference genes for qRT-PCR analyses in blueberry. RNA of high quality and yield was extracted from eight and six organs of rabbiteye and southern highbush blueberry, respectively, using a modified cetyltrimethyl ammonium bromide-based method. The expression stability of 12 reference genes was evaluated. UBIQUITIN-CONJUGATING ENZYME (UBC28), RNA HELICASE-LIKE (RH8), CLATHRIN ADAPTER COMPLEXES MEDIUM SUBUNIT FAMILY PROTEIN (CACSa), and POLYUBIQUITIN (UBQ3b) were the most stably expressed genes across multiple organs in both blueberry species. Further, the expression stability of the reference genes in the branch abscission zone following treatment with fruit abscission-inducing compounds was analyzed. CACSa, RH8, and UBC28 were the most stably expressed genes in the abscission zone under abscission-inducing conditions. We suggest a preliminary evaluation of UBC28, CACSa, RH8, and UBQ3b to identify the most suitable reference genes for the experimental conditions under consideration in blueberry.

The isolation of high quality RNA from the fruit of avocado ( Persea americana Mill.)

The fruit of avocado is high in structural carbohydrates and proteins and also contains polyphenols which interfere with the isolation of high quantity and quality of RNA. The aim of this study was to optimize RNA isolation from avocado fruit for subsequent complementary DNA (cDNA) transcription. We describe an optimized cetyltrimethylammonium bromide (CTAB)-based RNA extraction protocol that allows effective extraction of high-quality total RNA from the skin and flesh of the fruit without the use of phenol. Total RNA obtained from this procedure is of high quality and is undegraded, as assessed by spectrophotometric readings and electrophoresis, and was successfully used for cDNA synthesis. This is a first report of effective RNA extraction from avocado fruit.

A Protocol for Extraction of High-quality RNA and DNA from Peanut Plant Tissues

Peanuts are an increasingly important global food source. However, until recently the lack of effective protocols for the extraction of nucleic acids has made molecular studies of peanut development and maturation difficult. Here, we describe a method to isolate high-quality RNA and DNA from peanut tissue and have successfully applied this method to peanut plant roots, stems, leaves, flowers, and seeds. Spectrophotometric analysis showed that the average yields of total RNA from 100mg of peanut materials ranged from 24.52 to 74.6μg, and those of genomic DNA from the same tissues ranged from 23.47 to 57.68μg. Using this protocol, we obtained OD260/280 values between 1.9 and 2.0 and isolated RNA which could be reverse transcribed in a manner suitable for RT-qPCR and expression analysis. In addition, genomic DNA isolated using this method produced reliable restriction enzyme digestion patterns and could be used for Southern blot hybridization.

Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis

BackgroundMany experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction (real time qRT-PCR). Although several commercial kits are available for high-throughput RNA extraction in 96-well format, only one non-kit method has been described in the literature using the commercial reagent TRIZOL.ResultsWe describe an unusual phenomenon when using TRIZOL reagent with young Arabidopsis seedlings. This prompted us to develop a high-throughput RNA extraction protocol (HTP96) adapted from a well established phenol:chloroform-LiCl method (P:C-L) that is cheap, reliable and requires no specialist equipment. With this protocol 192 high quality RNA samples can be prepared in 96-well format in three hours (less than 1 minute per sample) with less than 1% loss of samples. We demonstrate that the RNA derived from this protocol is of high quality and suitable for use in real time qRT-PCR assays.ConclusionThe development of the HTP96 protocol has vastly increased our sample throughput, allowing us to fully exploit the large sample capacity of modern real time qRT-PCR thermocyclers, now commonplace in many labs, and develop an effective high-throughput gene expression platform. We propose that the HTP96 protocol will significantly benefit any plant scientist with the task of obtaining hundreds of high quality RNA extractions.

Optimization of the RNA extraction method for transcriptome studies of Salmonella inoculated on commercial raw chicken breast samples

BackgroundThere has been increased interest in the study of molecular survival mechanisms expressed by foodborne pathogens present on food surfaces. Determining genomic responses of these pathogens to antimicrobials is of particular interest since this helps to understand antimicrobial effects at the molecular level. Assessment of bacterial gene expression by transcriptomic analysis in response to these antimicrobials would aid prediction of the phenotypic behavior of the bacteria in the presence of antimicrobials. However, before transcriptional profiling approaches can be implemented routinely, it is important to develop an optimal method to consistently recover pathogens from the food surface and ensure optimal quality RNA so that the corresponding gene expression analysis represents the current response of the organism. Another consideration is to confirm that there is no interference from the "background" food or meat matrix that could mask the bacterial response.FindingsOur study involved developing a food model system using chicken breast meat inoculated with mid-log Salmonella cells. First, we tested the optimum number of Salmonella cells required on the poultry meat in order to extract high quality RNA. This was analyzed by inoculating 10-fold dilutions of Salmonella on the chicken samples followed by RNA extraction. Secondly, we tested the effect of two different bacterial cell recovery solutions namely 0.1% peptone water and RNAprotect (Qiagen Inc.) on the RNA yield and purity. In addition, we compared the efficiency of sonication and bead beater methods to break the cells for RNA extraction. To check chicken nucleic acid interference on downstream Salmonella microarray experiments both chicken and Salmonella cDNA labeled with different fluorescent dyes were mixed together and hybridized on a single Salmonella array. Results of this experiment did not show any cross-hybridization signal from the chicken nucleic acids. In addition, we demonstrated the application of this method in a meat model transcriptional profiling experiment by studying the transcriptomic response of Salmonella inoculated on chicken meat and exposed to d-limonene. We successfully applied our method in this experiment to recover the bacterial cells from the meat matrix and to extract the RNA. We obtained high yield and pure RNA. Subsequently, the RNA was used for downstream transcriptional profiling studies using microarrays and over 600 differentially regulated genes were identified.ConclusionsOur result showed that 8 log cfu/g of Salmonella is ideal to obtain optimal RNA amount and purity. Our results demonstrated that RNAprotect yielded higher RNA amounts (approximately 10 to 30 fold) when compared to 0.1% peptone water. The differences between the RNAprotect and 0.1% peptone samples were significant at a p-value of 0.03 for the bead beater method and 0.0005 for the sonication method, respectively. The microarray experiment demonstrated that the chicken samples do not interfere with the hybridization of Salmonella cDNA on the array slide. Hence, the background chicken RNA will not interfere with the microarray analysis when poultry meat models are used. Finally, we successfully demonstrated the application of the poultry meat model proposed in this study by conducting transcriptional profiling analysis of Salmonella inoculated on the poultry. Results of this study proved that this method has the potential to be employed in other meat model studies.

An integrated microfluidic system capable of sample pretreatment and hybridization for microarrays

Sample pretreatment is a critical series of processing steps in most protocols involving microarrays. It usually involves a labor-intensive, time-consuming process, which also requires bulky and costly apparatus to ensure a high-quality RNA extraction. Furthermore, an efficient hybridization process is also critical for practical applications in microarrays. The present study, therefore, reports a new microfluidic system capable of automatically performing the sample pretreatment and hybridization processes for microarrays. Process steps including cell lysis, extraction of messenger ribonucleic acid (mRNA), reverse transcription (RT), purification of complementary deoxyribonucleic acid (cDNA), and hybridization, are all performed automatically in this miniature system. When compared to the conventional methods, which usually require well-trained personnel and time consuming processes, this proposed microfluidic system can perform the entire experimental protocol automatically within 6 h. This developed system may provide a useful platform for subsequent genetic analysis and diagnostic applications.