A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer

Molecular cancer

2022 Mar 22

Fletcher, CE;Deng, L;Orafidiya, F;Yuan, W;Lorentzen, MPGS;Cyran, OW;Varela-Carver, A;Constantin, TA;Leach, DA;Dobbs, FM;Figueiredo, I;Gurel, B;Parkes, E;Bogdan, D;Pereira, RR;Zhao, SG;Neeb, A;Issa, F;Hester, J;Kudo, H;Liu, Y;Philippou, Y;Bristow, R;Knudsen, K;Bryant, RJ;Feng, FY;Reed, SH;Mills, IG;de Bono, J;Bevan, CL;
PMID: 35317841 | DOI: 10.1038/s44161-022-00042-8

miR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent.RNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq).miR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival. INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes. NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy.A balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.

GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

Science Translational Medicine

2019 Mar 27

Smith EL, Harrington K, Staehr M, Masakayan R, Jones J, Long TJ, Ng KY, Ghoddusi M, Purdon TJ, Wang X, Do T, Pham MT, Brown JM, De Larrea CF, Olson E, Peguero E, Wang P, Liu H, Xu Y, Garrett-Thomson SC, Almo SC, Wendel H-G, Riviere I, Liu C, Sather B and Brentjens RJ
| DOI: 10.1126/scitranslmed.aau7746

Chimeric antigen receptor (CAR) T cells, a type of cell-based immunotherapy, have shown some promising results in multiple myeloma, a bone marrow cancer. These earlier CAR T cells targeted a protein called B cell maturation antigen, but some patients’ cancer cells expressed little to none of this antigen and were therefore resistant to the CAR T cells. Smith et al. identified another target antigen for multiple myeloma, called GPRC5D. The authors demonstrated that CAR T cells against GPRC5D are effective in mouse models, even those with tumors that are resistant to the earlier CARs, and they are safe in mice and primates.Early clinical results of chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) for multiple myeloma (MM) appear promising, but relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating identification of additional targets. The orphan G protein–coupled receptor, class C group 5 member D (GPRC5D), normally expressed only in the hair follicle, was previously identified as expressed by mRNA in marrow aspirates from patients with MM, but confirmation of protein expression remained elusive. Using quantitative immunofluorescence, we determined that GPRC5D protein is expressed on CD138+ MM cells from primary marrow samples with a distribution that was similar to, but independent of, BCMA. Panning a human B cell–derived phage display library identified seven GPRC5D-specific single-chain variable fragments (scFvs). Incorporation of these into multiple CAR formats yielded 42 different constructs, which were screened for antigen-specific and antigen-independent (tonic) signaling using a Nur77-based reporter system. Nur77 reporter screen results were confirmed in vivo using a marrow-tropic MM xenograft in mice. CAR T cells incorporating GPRC5D-targeted scFv clone 109 eradicated MM and enabled long-term survival, including in a BCMA antigen escape model. GPRC5D(109) is specific for GPRC5D and resulted in MM cell line and primary MM cytotoxicity, cytokine release, and in vivo activity comparable to anti-BCMA CAR T cells. Murine and cynomolgus cross-reactive CAR T cells did not cause alopecia or other signs of GPRC5D-mediated toxicity in these species. Thus, GPRC5D(109) CAR T cell therapy shows potential for the treatment of advanced MM irrespective of previous BCMA-targeted therapy.

Automated Tumour Recognition and Digital Pathology Scoring Unravels New Role for PD-L1 in Predicting Good Outcome in ER-/HER2+ Breast Cancer.

Journal of Oncology (2018)

2018 Dec 17

Humphries MP, Hynes S, Bingham V, Cougot D, James J, Patel-Socha F, Parkes EE, Blayney JK, Rorke MA, Irwin GW, McArt DG, Kennedy RD, Mullan PB, McQuaid S, Salto-Tellez M, Buckley NE.
| DOI: 10.1155/2018/2937012

The role of PD-L1 as a prognostic and predictive biomarker is an area of great interest. However, there is a lack of consensus on how to deliver PD-L1 as a clinical biomarker. At the heart of this conundrum is the subjective scoring of PD-L1 IHC in most studies to date. Current standard scoring systems involve separation of epithelial and inflammatory cells and find clinical significance in different percentages of expression, e.g., above or below 1%. Clearly, an objective, reproducible and accurate approach to PD-L1 scoring would bring a degree of necessary consistency to this landscape. Using a systematic comparison of technologies and the application of QuPath, a digital pathology platform, we show that high PD-L1 expression is associated with improved clinical outcome in Triple Negative breast cancer in the context of standard of care (SoC) chemotherapy, consistent with previous findings. In addition, we demonstrate for the first time that high PD-L1 expression is also associated with better outcome in ER- disease as a whole including HER2+ breast cancer. We demonstrate the influence of antibody choice on quantification and clinical impact with the Ventana antibody (SP142) providing the most robust assay in our hands. Through sampling different regions of the tumour, we show that tumour rich regions display the greatest range of PD-L1 expression and this has the most clinical significance compared to stroma and lymphoid rich areas. Furthermore, we observe that both inflammatory and epithelial PD-L1 expression are associated with improved survival in the context of chemotherapy. Moreover, as seen with PD-L1 inhibitor studies, a low threshold of PD-L1 expression stratifies patient outcome. This emphasises the importance of using digital pathology and precise biomarker quantitation to achieve accurate and reproducible scores that can discriminate low PD-L1 expression.

Heterogeneity of Vascular Endothelial Growth Factor Receptors 1, 2, 3 in Primary Human Colorectal Carcinoma.

Anticancer Res.

2016 Jun 01

Nasir A, Reising LO, Nedderman DM, Fulford AD, Uhlik MT, Benjamin LE, Schade AE, Holzer TR.
PMID: 27272777 | DOI: -

Abstract

BACKGROUND:

The vascular endothelial growth factor (VEGF) pathway plays an important role in growth and progression of human cancer, including colorectal carcinomas (CRC). The key mediators of VEGF signaling are VEGFR1, VEGFR2, and VEGFR3, part of a family of related receptor tyrosine kinases. The relative expression, activity, or interplay among these receptors may determine the response of CRC patients to anti-angiogenic therapies.

MATERIALS AND METHODS:

We developed technically sound immunohistochemical (IHC) assays to quantify VEGFR1, 2 and 3, and using a well-annotated CRC tissue microarray (TMA), we carried out comprehensive comparative evaluation of the three VEGFRs in archival primary CRC tissues (n=84). For each TMA core, tumor cell VEGFR1 expression was reported as H-score (range=0-300); vascular VEGFR2/VEGFR3 expression was manually scored as the number of receptor-positive tumor stromal vessels. Each case was defined as VEGFR1/ VEGFR2/VEGFR3-negative, low, medium or high.

RESULTS:

Based on the differential expression of the three VEGFRs, eight VEGFR staining profiles were observed: Triple VEGFR positive (n=12, 14%), VEGFR1 predominant (n=17, 20%), VEGFR2 predominant (n=7, 8%), VEGFR3 predominant (n=1, 1%), VEGFR1/2 predominant (n=39, 46%), VEGFR1/3 predominant (n=2, 2%), VEGFR2/3 predominant (n=3, 4%), and triple-VEGFR-negative (n=3, 4%).

CONCLUSION:

Herein we demonstrated heterogeneity of expression of VEGFRs in human CRC stromal vessels and tumor cells. The observed VEGFR expression-based subsets of human CRCs may reflect differences in biology of pathologic angiogenesis in primary CRC tissues. Furthermore, the heterogeneity of expression of VEGFRs unraveled in this analysis merits independent validation in larger cohorts of primary and metastatic human CRC tissues and in pertinent experimental models treated with various anti-angiogenic therapies.

Fibronectin Produced by Cerebral Endothelial and Vascular Smooth Muscle Cells Contributes to Perivascular Extracellular Matrix in Late-Delayed Radiation-Induced Brain Injury.

Radiat Res.

2018 Jul 17

Andrews RN, Caudell DL, Metheny-Barlow LJ, Peiffer AM, Tooze JA, Bourland JD, Hampson RE, Deadwyler SA, Cline JM.
PMID: 30016219 | DOI: 10.1667/RR14961.1

Late-delayed radiation-induced brain injury (RIBI) is a major adverse effect of fractionated whole-brain irradiation (fWBI). Characterized by progressive cognitive dysfunction, and associated cerebrovascular and white matter injury, RIBI deleteriously affects quality of life for cancer patients. Despite extensive morphological characterization of the injury, the pathogenesis is unclear, thus limiting the development of effective therapeutics. We previously reported that RIBI is associated with increased gene expression of the extracellular matrix (ECM) protein fibronectin (FN1). We hypothesized that fibronectin contributes to perivascular ECM, which may impair diffusion to the dependent parenchyma, thus contributing to the observed cognitive decline. The goal of this study was to determine the localization of fibronectin in RIBI and further characterize the composition of perivascular ECM, as well as identify the cell of origin for FN1 by in situ hybridization. Briefly, fibronectin localized to the vascular basement membrane of morphologically normal blood vessels from control comparators and animals receiving fWBI, and to the perivascular space of edematous and fibrotic vascular phenotypes of animals receiving fWBI. Additional mild diffuse parenchymal staining in areas of vascular injury suggested blood-brain-barrier disruption and plasma fibronectin extravasation. Perivascular ECM lacked amyloid and contained lesser amounts of collagens I and IV, which localized to the basement membrane. These changes occurred in the absence of alterations in microvascular area fraction or microvessel density. Fibronectin transcripts were rarely expressed in control comparators, and were most strongly induced within cerebrovascular endothelial and vascular smooth muscle cells after fWBI. Our results demonstrate that fibronectin is produced by cerebrovascular endothelial and smooth muscle cells in late-delayed RIBI and contributes to perivascular ECM, which we postulate may contribute to diffusion barrier formation. We propose that pathways that antagonize fibronectin deposition and matrix assembly or enhance degradation may serve as potential therapeutic targets in RIBI.

Composition and Clinical Impact of the Immunologic Tumor Microenvironment in Oral Squamous Cell Carcinoma.

J Immunol. 2018 Dec 10.

2018 Dec 10

Boxberg M, Leising L, Steiger K, Jesinghaus M, Alkhamas A, Mielke M, Pfarr N, Götz C, Wolff KD, Weichert W, Kolk A.
PMID: 30530592 | DOI: 10.4049/jimmunol.1800242

Immunotherapy shows promising results and revolutionizes treatment of oral squamous cell carcinoma (OSCC). The immunologic microenvironment might have prognostic/predictive implications. Morphologic immunologic parameters (inflammatory infiltrate, stromal content, and budding activity [BA] [potentially indicating epithelial–mesenchymal transition]) were evaluated in 66 human primary therapy-naive OSCCs. Intraepithelial/stromal tumor-infiltrating lymphocytes (TILs; CD3+/CD4+/CD8+/CD4+FOXP3+/IL-17A+) were quantified, and ratios were calculated. HLA class I in tumor cells was evaluated immunohistochemically. mRNA in situ hybridization to detect IFN-γ was performed. Analysis was performed within invasive front (IF) and tumor center (TCe). Decreased HLA expression was associated with low TIL density, pronounced stromal content, and high BA; IFN-γ in TILs was correlated with high-density TILs; and IFN-γ in tumor cells was correlated with absence of BA (p < 0.05). Heterogeneity of parameters (TCe/IF) was rare. Low density of stromal CD4+FOXP3+ TILs within TCe and IF was identified as an independent prognostic factor for poor overall, disease-specific, and disease-free survival (p ≤ 0.011). Refining prognostication in OSCC with high-density CD4+FOXP3+ infiltrate within TCe and/or IF, high FOXP3:CD4 ratio was significantly correlated with favorable outcome in this subgroup. Furthermore, high-stromal CD8:CD4 ratio was found to be an independent favorable prognostic factor. In summary, immunologic parameters were closely intertwined. Morphologic correlates of epithelial–mesenchymal transition were associated with downregulation of HLA and decreased inflammation. Heterogeneity was infrequent. Low-density stromal CD4+FOXP3+ infiltrate within TCe and IF was an independent poor prognostic factor. Stratification of cases with high-density CD4+FOXP3+ TILs by FOXP3:CD4 ratio enables refinement of prognostication of this subgroup. CD8:CD4 ratio was identified as an independent prognostic factor.

Critical appraisal of PD-L1 reflex diagnostic testing: current standards and future opportunities.

J Thorac Oncol. 2018 Oct 5.

2018 Oct 05

Humphries MP, McQuaid S, Craig S, Bingham V, Maxwell P, Maurya M, McLean F, Sampson J, Higgins P, Greene C, James J, Salto-Tellez M.
PMID: 30296485 | DOI: 10.1016/j.jtho.2018.09.025

Abstract INTRODUCTION: Patient suitability to anti-PD-L1 immune checkpoint inhibition is key to the treatment of non-small cell lung cancer (NSCLC). We present, applied to PD-L1 testing: a comprehensive cross-validation of two immunohistochemistry (IHC) clones; our descriptive experience in diagnostic reflex testing; the concordance of IHC to in-situ RNA (RNA-ISH); and application of digital pathology. METHODS: 813 NSCLC tumour samples collected from 564 diagnostic samples were analysed prospectively and 249 diagnostic samples analysed retrospectively in TMA format. Validated methods for IHC and RNA-ISH were tested in TMAs and full sections and the QuPath system used for digital pathology analysis. RESULTS: Antibody concordance of clones SP263 and 22C3 validation was 97-98% in squamous cell carcinoma and adenocarcinomas, respectively. Clinical NSCLC cases were reported as PD-L1 negative (48%), 1-49% (23%) and >50% (29%), with differences associated to tissue-type and EGFR status. Comparison of IHC and RNA-ISH was highly concordant in both subgroups. Comparison of digital assessment versus manual assessment was highly concordant. Discrepancies were mostly around the 1% clinical threshold. Challenging IHC interpretation included a) calculating the total tumour cell denominator and the nature of PD-L1 expressing cell aggregates in cytology samples; b) peritumoral expression of positive immune cells; c) calculation of positive tumour percentages around clinical thresholds; d) relevance of the 100 malignant cell rule. CONCLUSIONS: Sample type and EGFR status dictate differences in the expected percentage of PD-L1 expression. Analysis of PD-L1 is challenging, and interpretative guidelines are discussed. PD-L1 evaluation by RNA-ISH and digital pathology appear reliable, particularly in adenocarcinomas.

RNA Chromogenic in situ Hybridization Assay with Clinical Automated Platform is a Sensitive Method in Detecting High-risk Human Papillomavirus in Squamous Cell Carcinoma

Human Pathology

2017 Mar 14

Mendez-Pena JE, Sadow PM, Vania Nose VN, Hoang MP.
PMID: 28302536 | DOI: 10.1016/j.humpath.2017.02.021

Detection of active human papillomavirus (HPV) is clinically important, as its presence has been shown to correlate with favorable clinical outcomes and better response to treatment in oropharyngeal squamous cell carcinomas (SCC). Using a clinical automated platform, we compared the performance of commercially available HPV DNA and RNA in situ hybridization (ISH) probes in archival tissues of 57 SCC. Importantly, a clinical automated platform gives 1) consistent and reproducible results for HPV ISH and 2) better standardization across clinical laboratories. Compared to polymerase chain reaction (PCR) results, RNA ISH exhibited 93% concordance versus 81% of DNA ISH. RNA ISH was more sensitive than DNA ISH (100% versus 88%), and more specific (87% versus 74%). When only accounting for 2-3+ positivity, sensitivity was 92% for RNA ISH versus 73% for DNA ISH, highlighting the ease of interpretation. p16 exhibited 96% sensitivity while specificity was only 55%. In 3 cases both RNA and DNA ISH were positive while PCR results were negative, suggesting that ISH methods might be a more sensitive method. Performing on a clinical automated platform, RNA ISH is sensitive in determining high-risk HPV status in formalin-fixed paraffin-embedded tissues and has the potential of being a standalone clinical test.

Automated RNA In Situ Hybridization for 18 High Risk Human Papilloma Viruses in Squamous Cell Carcinoma of the Head and Neck: Comparison With p16 Immunohistochemistry.

Appl Immunohistochem Mol Morphol.

2017 Aug 02

Drumheller B, Cohen C, Lawson D, Siddiqui MT.
PMID: 28777152 | DOI: 10.1097/PAI.0000000000000550

Detection of human papilloma virus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is important, as HPV-associated HNSCCs respond better to therapy. The RNAscope HPV-test is a novel RNA in situ hybridization (ISH) technique which strongly stains transcripts of E6 and E7 mRNA in formalin-fixed, paraffin-embedded tissue, with the potential to replace the indirect immunohistochemical (IHC) marker for p16 protein. A direct clinical comparison between p16 IHC and an automated RNA ISH using 18 probes has not been established. Samples from 27 formalin-fixed, paraffin-embedded HNSCC cases from the Emory University Hospital archives were stained using 18 individual RNA ISH probes for high-risk HPV (RNAscope 2.5 LS Probe ) on a Leica autostainer (Buffalo Grove, IL) and were compared with p16 IHC. Two pathologists reviewed and reached a consensus on all interpretations. The RNAscope technique was positive in 89% (24/27) and the p16 IHC was positive in 78% (21/27). The RNAscope was negative in 11.1% of samples (3/27) and the p16 IHC-negative in 22.2% (6/27). The RNA ISH detected 100% of the p16-positive IHC-stained slides and had a concordance of 88.9% (24/27). This easy to interpret automated staining method for 18 high-risk HPV genotypes is a feasible replacement for the indirect p16 IHC method.

Albumin In Situ Hybridization Can Be Positive in Adenocarcinomas and Other Tumors From Diverse Sites

Am J Clin Pathol

2019 May 20

Nasir A, Lehrke HD, Mounajjed T, Said S, Zhang L, Yasir S, Shah SS, Chandan VS, Smyrk TC, Moreira RK, Boland Froemming JM, Herrera Hernandez LP, Wu TT, Graham RP.
PMID: 31107526 | DOI: 10.1093/ajcp/aqz032

OBJECTIVES:

Albumin messenger RNA (mRNA) expression is a marker of hepatocellular differentiation. Most published data are from review of tissue microarrays, and albumin in situ hybridization (ISH) expression across several tumor types is incompletely characterized.

METHODS:

Sections from 221 tumors were evaluated for albumin mRNA. Immunohistochemistry was used to confirm diagnoses. Albumin ISH was performed according to manufacturer-provided instructions. Fifty-nine cases were evaluated with both commercial ISH assays.

RESULTS:

Albumin mRNA was detected in all hepatocellular carcinomas (HCCs) and 81% of intrahepatic cholangiocarcinomas. Lung (20%), gallbladder (39%), hepatoid pancreatic (n = 1 of 1) adenocarcinoma, breast invasive ductal carcinoma (18%), yolk sac tumor (25%), and acinar cell carcinoma (29%) showed expression. Both assays were concordant in 93% of cases.

CONCLUSIONS:

Albumin ISH was expressed in all HCCs studied. It was also positive in intrahepatic cholangiocarcinoma and patchy positive in gallbladder adenocarcinoma and a subset of other neoplasms, which can be a potential pitfall

Nav1.7 is essential for nociceptor action potentials in the mouse in a manner independent of endogenous opioids

Neuron

2023 Jun 15

Deng, L;Dourado, M;Reese, RM;Huang, K;Shields, SD;Stark, KL;Maksymetz, J;Lin, H;Kaminker, JS;Jung, M;Foreman, O;Tao, J;Ngu, H;Joseph, V;Roose-Girma, M;Tam, L;Lardell, S;Orrhult, LS;Karila, P;Allard, J;Hackos, DH;
PMID: 37352856 | DOI: 10.1016/j.neuron.2023.05.024

Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overexpression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low-threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and in vivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action potentials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.

HPV Virus Transcriptional Status Assessment in a Case of Sinonasal Carcinoma.

Int J Mol Sci.

2018 Mar 16

Ilardi G, Russo D, Varricchio S, Salzano G, Dell'Aversana Orabona G, Napolitano V, Di Crescenzo RM, Borzillo A, Martino F, Merolla F, Mascolo M, Staibano S.
PMID: 29547549 | DOI: 10.3390/ijms19030883

Human Papilloma Virus (HPV) can play a causative role in the development of sinonasal tract malignancies. In fact, HPV may be the most significant causative agent implicated in sinonasal tumorigenesis and is implicated in as many as 21% of sinonasal carcinomas. To date, there are no definitive, reliable and cost-effective, diagnostic tests approved by the FDA for the unequivocal determination of HPV status in head and neck cancers. We followed an exhaustive algorithm to correctly test HPV infection, including a sequential approach with p16INK4a IHC, viral DNA genotyping and in situ hybridization for E6/E7 mRNA. Here, we report a case of sinonasal carcinoma with discordant results using HPV test assays. The tumor we describe showed an irregular immunoreactivity for p16INK4a, and it tested positive for HPV DNA; nevertheless, it was negative for HR-HPV mRNA. We discuss the possible meaning of this discrepancy. It would be advisable to test HPV transcriptional status of sinonasal carcinoma on a diagnostic routine basis, not only by p16INK4a IHC assay, but also by HPV DNA genotyping and HR-HPV mRNA assessment.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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Abstract

BACKGROUND:

MATERIALS AND METHODS:

RESULTS:

CONCLUSION:

OBJECTIVES:

METHODS:

RESULTS:

CONCLUSIONS:

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Advanced Cell Diagnostics

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