Mean RNA Integrity Number Research Articles

Extraction of high-quality RNA from mouse pancreatic tumors

Extraction of high-quality RNA from pancreatic tumors for sequencing purposes is technically challenging, as the pancreas is an organ rich in ribonucleases. The majority of the established RNA isolation protocols for use with primary pancreatic tissue involve perfusion of RNA stabilizing reagent into the pancreatic tissue to protect RNA integrity before extraction. However, the additional time needed for this procedure can actually lead to further RNA degradation. We optimized a protocol suitable for high quality RNA isolation from mouse pancreatic tumors that is a simple, fast, and inexpensive modification of existing methods, combining the use of liquid nitrogen and guanidinium thiocyanate-chloroform extraction. Through this procedure, the mean RNA Integrity Number value obtained for RNA isolated from pancreatic tumors was 9.0, and was reproducibly suitable for RNAseq and qPCR.•a protocol suitable for high quality RNA isolation from mouse pancreatic tumors as well as normal pancreas•combining the use of liquid nitrogen and guanidinium thiocyanate-chloroform extraction

A method of processing nasopharyngeal swabs to enable multiple testing.

ObjectiveTo develop a method to perform multiple tests on single nasopharyngeal (NP) swab.Study designWe collected a NP swab on children aged 2–12 years with acute sinusitis and processed it for bacterial culture, viruses, cytokine expression, and 16S ribosomal RNA gene sequencing analysis. During the course of the study, we expand the scope of evaluation to include RNA sequencing, which we accomplished by cutting the tip of the swab.ResultsOf the 174 children enrolled, 126 (72.4%) had a positive bacterial culture and 121(69.5%) tested positive for a virus. Cytokine measurement, as judged by the adequate levels of a housekeeping enzyme (GAPDH), appeared successful. From the samples used for 16S ribosomal sequencing we recovered, on average, 16,000 sequences per sample, accounting for a total of 2,646 operational taxonomic units across all samples sequenced. Samples used for RNA sequencing had a mean RNA Integrity number of 6.0. Cutting the tip of the swab did not affect the recovery yield for viruses or bacteria, nor did it affect species richness in microbiome analysis.ConclusionWe describe a minimally invasive sample collection protocol that allows for multiple diagnostic and research investigations in young children.

Improved method for isolating high-quality RNA from mouse bone with RNAlater at room temperature

Accurate gene expression analysis of bone requires the ability to isolate RNA of good quality. Isolation of intact RNA from frozen bone tissue is problematic since RNA rapidly becomes degraded after thawing. Since we are interested in assessing gene expression from both bone marrow and mineralized bone, we aimed to develop improved simple, robust and statistically validated methods providing high-quality RNA from both mouse femur shaft and femur marrow. RNA integrity was quantified by the RNA Integrity Number (RIN) measured on a TapeStation. While the RNA stabilization reagent RNAlater is not commonly used or recommended for mineralized bone, we found that preservation methods with RNAlater significantly improved the RNA quality with a mean RIN for the femur shaft of 8.0 and a mean RIN for femur marrow of 9.6. With RNAlater, high quality RNA with a mean RIN of 9.3 could also be isolated from lumbar vertebral bone. A further advantage of using RNAlater is that the tissue can be allowed to thaw to room temperature before TRI Reagent lysis without any loss of RNA integrity. A comparison of the TRI Reagent method with a hybrid method combining TRI Reagent lysis with RNeasy column purification showed no difference in RNA integrity. However, the hybrid method seemed to give femur shaft RNA with fewer impurities inhibiting qRT-PCR.

Temporal RNA Integrity Analysis of Archived Spaceflight Biological Samples

Abstract In spaceflight experiments, model organisms are used to assess the effects of microgravity on specific biological systems. In many cases, only one biological system is of interest to the Principal Investigator. To maximize the scientific return of experiments, the remaining spaceflight tissue is categorized, documented, and stored in the biobank at NASA Ames Research Center, which is maintained by the Ames Life Science Data Archive (ALSDA). The purpose of this study is to evaluate the state of a sample set of tissues from the ALSDA biobank. Garnering information – such as downstream functional analysis for the generation of omics datasets – from tissues is, in part, dependent on the state of sample preservation. RNA integrity number (RIN) values have been calculated for rodent liver tissues that were part of scientific payloads returned from the International Space Station (ISS). Rat livers from Spacelab Life Sciences 1 (SLS-1) and mouse livers from Commercial Biomedical Test Module 3 (CBTM-3), Rodent Research 1 (RR1), and Rodent Research 3 (RR3) were tested. It was found that mean RIN values from CBTM-3, RR1, and RR3 were suitable for downstream functional analysis (RIN > 5) while the mean RIN value for SLS-1 was not (RIN = 2.5 ± 0.1). Information from this study lays the foundation for future efforts in determining the types of assays that are most appropriate for different tissues in the ALSDA biobank and similar preservation facilities, which would aid in shaping the design of experiments.

Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays

AimsHistopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS)...

Abstract B39: A pilot study to establish procedures for DNA and RNA isolation from African esophageal tumor specimens

Abstract Objective: We propose to utilize tumor biospecimens from patients with esophageal squamous cell carcinoma (ESCC) to identify possible genetic, molecular, and infectious determinants of this high-incidence disease in East Africa. However, laboratory technologies in East Africa are not available to support DNA and RNA extraction and genome and transcriptome sequencing from patient specimens. In preparation for establishment of a biorepository for African ESCC specimens, we compared different fixation and preservation media for transportability over long distances and success in preserving the genetic integrity and expression profiles of ESCC. Methods: Patients with a suspected diagnosis of ESCC at Muhimbili National Hospital in Dar es Salaam, Tanzania were identified and consented prior to endoscopic evaluations. For patients with endoscopic findings consistent with ESCC, tumor biopsies were obtained and stored using two different fixation and preservation media: PAXgene® Tissue Container (n=2) and RNAlater® (n=2). All specimens were shipped at room temperature from Dar es Salaam to San Francisco. DNA and RNA quantity was measured by nanodrop method and DNA was further confirmed by picogreen method, yielding measures of total DNA and RNA acquired (ug). DNA quality was measured as percent of total DNA degradation to between 200-1000 bp. RNA quality measure was determined by bioanalyzer output measure RNA Integrity Number (RIN). Results: Specimens for our first 10 patients were analyzed. Average transit time of biopsy specimens in preservative at room temperature was 6.2 days (range 3.5-9.0). Tumor specimens preserved with PAXgene® yielded a mean of 7.7 ug total DNA and 8.6 ug total RNA. Tumor specimens preserved with RNAlater® yielded a mean 1.4 ug of DNA and 8.2 ug of RNA. DNA degradation products were 0% of total with PAXgene® versus 6% of total with RNAlater®. Specimens preserved with PAXgene® yielded a mean RIN of 4, while RNAlater® yielded a mean RIN of 9. DNA and RNA quality were not associated with length of time at room temperature, up to a maximum of 9 days. Conclusion: Tissue preserved using the PAXgene® Tissue Container yielded higher DNA quantity and quality than tissue preserved in RNAlater®. RNA quantity was comparable for both mediums, but RNAlater® resulted in superior RNA quality versus PAXgene®. Both mediums allowed for flexible transport of specimens at room temperature and merit further inquiry into their potential as cost-effective methods to facilitate molecular analyses for geographically isolated diseases in Africa. Based upon our pilot data, each medium offers unique advantages. Our expanded study will continue to utilize both mediums to optimize isolation of both DNA and RNA. We will plan to present data for an enriched sample size. Funding source: National Institutes of Health, National Cancer Institute Cancer Center Administrative Supplement to Promote Cancer Prevention and Control Research in Low and Middle Income Countries, A119617, [CA-0082629]. Citation Format: Beatrice Paul Mushi, John Greer, Charles William Cahalane, II, Msiba Selekwa, Ali Mwanga, Larry Akoko, Elia Mmbaga, Eric Collisson, Katherine Van Loon. A pilot study to establish procedures for DNA and RNA isolation from African esophageal tumor specimens [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B39.

Abstract A70: Identification of pancreatic cancer neoantigens by exome and RNA sequencing analysis

Abstract Introduction: Recent reports suggest that the mutational landscape predicts response to checkpoint blockade therapy in non-small cell lung cancer and colorectal cancer. Neoantigens, identified by exome and RNA sequencing using immune prediction algorithms, have been successfully targeted by dendric cell vaccination in melanoma. The relative abundance of non-malignant cells within the stroma has made Identification of neoantigens in pancreatic cancer (PC) challenging. Here we report successful isolation and high quality sequencing of PC neoplastic ductal cells specifically, resulting in the identification of immunogenic mutations. Methods: Specimens from freshly resected PC tumors were collected in the operating room and immediately frozen in OCT. Laser microdissection was performed by a pathologist to isolate PC malignant cells from the stromal compartment at >90% purity. DNA and RNA were isolated from the purified tumors with nonsynonymous mutations identified by tumor/normal exome sequencing and expression verified by RNA sequencing. HLA typing was inferred from exome sequencing and verified by standard HLA typing techniques. Candidate neoantigen binding scores were determined via NetMHC. Tumor infiltrating lymphocyte (TIL) culture was carried out over 12-14 days by serial reduction of tumor concentration as the TIL proliferated in the presence of IL-2 containing media (50 units/mL). Results: Patient #567 was confirmed to express HLA-A3 and HLA-A23. 105 nonsynonymous mutations were identified by tumor/normal exome sequencing, all of which were single nucleotide polymorphisms. Candidate neoantigens were identified and prioritized based on predicted binding of the mutant amino acid sequence to MHC class I. 30 candidate neoantigens were prioritized for further evaluation based on a binding score of < 500 nM, and superior predicted binding associated with the mutant sequence compared to the wildtype sequence. Mutant allele expression was verified by a fragments per kilobase of exon per million fragments mapped (FKPM) >1, with a cut off for RNA variant allele frequency (VAF) greater than 20%. Using this stringent criteria 10 candidate epitopes were identified. Next, we validated this process in 17 consecutive PC patients. DNA and RNA were successfully isolated from 13 patients (76.5%), the remaining four had no viable tumor at pathology. The mean RNA integrity number (RIN) for the isolated samples was 7.6 (Range 5-8.7). Additionally, to test for existing immune response to the predicted epitopes TIL were successfully cultured from 6 of 8 (75%) attempted patients with a mean yield of 3.0x106 (Range 1x105 – 7.2x106). Conclusion: Obtaining high quality, reliable sequencing data from PC patients is possible using rigorous tumor purification techniques to specifically select malignant cells. Candidate neoantigens can be identified using a combination of epitope prediction algorithms. Expansion of TIL populations in vitro may offer functional studies to further refine the optimal selection of immunogenic neoantigens and optimize personalized vaccination strategies in PC. Citation Format: Darren R. Cullinan, S. Peter Goedegebuure, Chris A. Miller, Timothy M. Nywening, Samuel Kim, Elaine R. Mardis, William G. Hawkins, William E. Gillanders.{Authors}. Identification of pancreatic cancer neoantigens by exome and RNA sequencing analysis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A70.

Effects of schizophrenia risk variation in the NRG1 gene on NRG1-IV splicing during fetal and early postnatal human neocortical development.

Neuregulin 1 (NRG1) is a multifunctional neurotrophin that mediates neurodevelopment and schizophrenia risk. The NRG1 gene undergoes extensive alternative splicing, and association of brain NRG1 type IV isoform expression with the schizophrenia-risk polymorphism rs6994992 is a potential mechanism of risk. Novel splice variants of NRG1-IV (NRG1-IVNV), with predicted unique signaling capabilities, have been cloned in fetal brain tissue. The authors investigated the temporal dynamics of transcription of NRG1-IVNV, compared with the major NRG1 isoforms, across human prenatal and postnatal prefrontal cortical development, and they examined the association of rs6994992 with NRG1-IVNV expression. NRG1 type I-IV and NRG1-IVNV isoforms were evaluated with quantitative real-time polymerase chain reaction in human postmortem prefrontal cortex tissue samples at 14 to 39 weeks gestation and postnatal ages 0-83 years. The association of rs6994992 genotype with NRG1-IVNV expression and the subcellular distribution and proteolytic processing of NRG1-IVNV isoforms were also determined. Expression of NRG1 types I, II, and III was temporally regulated during prenatal and postnatal neocortical development. NRG1-IVNV was expressed from 16 weeks gestation until age 3. Homozygosity for the schizophrenia risk allele (T) of rs6994992 conferred lower cortical NRG1-IVNV levels. Assays showed that NRG1-IVNV is a novel nuclear-enriched, truncated NRG1 protein resistant to proteolytic processing. To the authors' knowledge, this study provides the first quantitative map of NRG1 isoform expression during human neocortical development and aging. It identifies a potential mechanism of early developmental risk for schizophrenia at the NRG1 locus, involving a novel class of NRG1 proteins.

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.

Changes in Gene Transcription Underlying the Aberrant Citrate and Choline Metabolism in Human Prostate Cancer Samples

Low concentrations of citrate and high concentrations of choline-containing compounds (ChoCC) are metabolic characteristics observed by magnetic resonance spectroscopy of prostate cancer tissue. The objective was to investigate the gene expression changes underlying these metabolic aberrations to find regulatory genes with potential for targeted therapies. Fresh frozen samples (n = 133) from 41 patients undergoing radical prostatectomy were included. Histopathologic evaluation was carried out for each sample before a metabolic profile was obtained with high-resolution magic angle spinning (HR-MAS) spectroscopy. Following the HR-MAS, RNA was extracted from the same sample and quality controlled before carrying out microarray gene expression profiling. A partial least square statistical model was used to integrate the data sets to identify genes whose expression show significant covariance with citrate and ChoCC levels. Samples were classified as benign, n = 35; cancer of low grade (Gleason score 6), n = 24; intermediate grade (Gleason score 7), n = 41; or high grade (Gleason score ≥ 8), n = 33. RNA quality was high with a mean RNA Integrity Number score of 9.1 (SD 1.2). Gene products predicting significantly a reduced citrate level were acetyl citrate lyase (ACLY, P = 0.003) and m-aconitase (ACON, P < 0.001). The two genes whose expression most closely accompanied the increase in ChoCC were those of phospholipase A2 group VII (PLA2G7, P < 0.001) and choline kinase α (CHKA, P = 0.002). By integrating histologic, transcriptomic, and metabolic data, our study has contributed to an expanded understanding of the mechanisms underlying aberrant citrate and ChoCC levels in prostate cancer.

Expression Analysis on Archival Material

Formalin-fixed paraffin-embedded (FFPE) tissue is the most common tissue specimen widely available. Moreover, long clinical follow-up is on hand. Therefore, FFPE material is a precious source of material for identifying predictive and/or prognostic biomarkers in cancer research on the basis of gene expression. However, the main drawback of FFPE tissue is the significant reduction in quantity and quality of the extracted RNA. The aim of this study is the comparison of different commercially available kits for the RNA isolation in FFPE tissue material. Five commercially available RNA isolation kits were tested and the concentration, purity, integrity, and raw cycle threshold values were determined. The mean total RNA concentrations were as follows: Qiagen 25957±19417 ng, Ambion 8249±2898 ng, SA Biosciences 8070±3700 ng, and Macherey-Nagel 622±394 ng. The mean A260/A280 ratios were as follows: Qiagen: 1.81, SA Biosciences: 0.66, Ambion: 1.03, and Macherey-Nagel: 1.04. The mean A260/A230 ratios were as follows: Qiagen: 1.88, SA Biosciences: 1.61, Ambion: 1.54, and Macherey-Nagel: 1.88. The RNA extractions from Epicentre could not be measured by the Nanodrop and, therefore, were excluded from further analysis. The mean RNA integrity number (range, 2.09 to 2.47) and the mean raw cycle threshold values (range, 33.43 to 35.37) were more or less the same for all the tested RNA isolation kits. Altogether, on the basis of the number of adequate isolations, the kit from Qiagen seems to be the most appropriate kit to be used in our further studies that require RNA isolation from FFPE material.

Variation of Peripheral Blood Mononuclear Cell RNA Quality in Archived Samples.

The Infectious Diseases BioBank (IDB) has consistently archived peripheral blood mononuclear cell (PBMNC) RNA for transcriptome analyses. RNA is particularly labile, and hence, these samples provide a sensitive indicator for assessing the IDB's quality-assurance measures. Independent analyses of 104 PBMNC RNA specimens from 26 volunteers revealed that the mean RNA integrity number (RIN) was high (9.02), although RIN ranged between scores of 7 and 10. This variation of RIN values was not associated with ischemic time, PBMNC quality, number of samples processed per day, self-medication after immunization, freezer location, donor characteristics, differential white blood cell counts, or daily variation in RNA extractions (all P>0.05). RIN values were related to the date of collection, with those processed during mid-summer having highest RIN scores (P=0.0001). Amongst specimens with the lowest RIN scores, no common feature could be identified. Thus, no technical explanation for the variation in RNA quality could be ascertained and these may represent normal physiological variations. These data provide strong evidence that current IDB protocols for the isolation and preservation PBMNC RNA are robust.

Extraction of RNA Using Fine-Needle Aspiration Samples Stored Under Different Conditions

: Diagnosis of lung cancer is achieved by fine-needle aspiration (FNA) techniques such as computed tomography-guided FNA and transbronchial needle aspiration. The purpose of this study was to establish an optimal storing method for molecular testing in FNA samples. : We performed FNA using a 21-gauge needle in surgically resected lung cancer samples. The aspirates were stored according to the following protocol: in group 1, the aspirate was snap frozen with liquid nitrogen and in group 2, the aspirate was mixed with RNA later. After sample collection from both groups, these samples were stored at -80°C for 6 months. RNA was extracted from each sample using a commercially available RNA extraction kit, and the quality and quantity of RNA were measured. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed for human actin β (hACTB) and keratin 19 (KRT19). : FNA was performed from 7 lung cancers. RNA was extracted from all samples. The median total amount of extracted RNA was 33.9 μg for group 1 and 35.8 μg for group 2. The mean RNA integrity number was 3.5 for group 1 and 6.3 for group 2. RT-PCR for hACTB and KRT19 could be successfully performed in all samples; however, the relative gene expression value showed intrasample variation. : Samples obtained from computed tomography-guided FNA or transbronchial needle aspiration may be used for genetic profiling of lung cancer. RNA can be extracted from FNA samples and can be used for RT-PCR. RNA quality may affect mRNA expression analysis; therefore, an optimal FNA sample storing protocol is essential.

A new method to provide a fresh frozen prostate slice suitable for gene expression study and MR spectroscopy

Fresh frozen tissue from radical prostatectomy specimens is highly valuable material for research on gene expression and cellular metabolites. The purpose of this study was to develop a standardized method to provide a representative high quality research sample from radical prostatectomy specimens without interfering with the routine histopathological procedure. A complete transversal slice is collected and snap-frozen before formalin fixation and routine processing of the remaining gland. The freezing preserves the original geometric shape, thus allowing subsampling of specific cell populations without thawing. RNA was extracted from 53 cylindrical subsamples (diameter 3 mm, thickness 2 mm) from 16 consecutive frozen slices. The histological pattern was evaluated by microscopy of a cryosection from sample before further analysis. Using this novel harvesting method close to 400 slices have been collected. Whenever tumor was present in both adjacent surrounding hematoxylin-eosin sections, we found cancer in 88% of the frozen slices. The extracted RNA showed very high quality with a mean RNA integrity number of 9.16 (SD 0.53). The MR spectra showed metabolic profiles containing several resonances, which deserve further evaluation as possible biomarkers for prostate cancer. After MR analysis the RNA was still highly intact with a mean RNA integrity number of 8.40 (SD 1.53), which makes it possible to correlate transcriptomic and metabolomic profiles of the extracted samples. We present a safe and standardized method for procurement of a high quality fresh frozen prostate slice, suitable for gene expression analysis and MR spectroscopy.

In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

BackgroundHousekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes.ResultsThe expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP) and peptidylprolyl isomerase A (PPIA), all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel.ConclusionOur study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.