Each anesthetized rat was secured on its dorsal side and a thorough sterilization of its ventral side using 70% alcohol was performed before exposing the abdominal viscera by a longitudinal ventral incision. The liver was flipped over and pushed aside; the bile duct (BD, Franklin Lake, NJ, USA) was located and traced to its joining point with the pancreatic duct (hepatopancreatic duct [HPD], Figure 1 A). Further, a 1-mL syringe (BD, Franklin Lake, NJ, USA) with a 30-G needle (BD, Franklin Lake, NJ, USA) (bent to 45° angle) was inserted into the lumen of the BD and the pancreas infused with RNAlater (Qiagen, Hilden, Germany) (Figure 1 A). To ensure complete perfusion, the HPD was occluded with a clamp just above the region where it joins the duodenum (sphincter of Oddi). In addition, forceps were used to hold and seal the other end of the BD to prevent retrograde perfusion of the liver (Figure 1 A,B). The location of the RNAlater (Qiagen) infusion site and clamping of the sphincter of Oddi were performed as previously described [4 (link)] and resulted in good perfusion, as indicated by the swelling of the pancreas (Figure 1 B). Prior to administering RNAlater (Qiagen), the perfusion technique was tested by infusing India ink (Loba Chemie, Colaba, India), which showed a very good distribution in the pancreas (Figure 1 C).
Rnalater
Manufactured by Qiagen
Sourced in Germany, United States, Spain, United Kingdom, Netherlands, Canada, Japan, Switzerland, France, Australia, Sweden, Belgium, Italy
RNAlater is a stabilization solution designed to rapidly permeate tissues and cells to stabilize and protect cellular RNA immediately after sample collection. It allows samples to be stored at ambient temperature for extended periods without degradation of RNA.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (179 mentions)
Trizol reagent, by Thermo Fisher Scientific (62 mentions)
Trizol, by Thermo Fisher Scientific (54 mentions)
Rneasy kit, by Qiagen (36 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (33 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (31 mentions)
Rneasy plus mini kit, by Qiagen (29 mentions)
Iscript cdna synthesis kit, by Bio-Rad (25 mentions)
2100 bioanalyzer, by Agilent Technologies (24 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (24 mentions)
Quantitect reverse transcription kit, by Qiagen (23 mentions)
Rneasy micro kit, by Qiagen (22 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (20 mentions)
Rlt buffer, by Qiagen (20 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (20 mentions)
Tissuelyser, by Qiagen (19 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (19 mentions)
Nanodrop, by Thermo Fisher Scientific (19 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (18 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (17 mentions)
1 499 protocols using rnalater
1
Perfusion of Rat Pancreas with RNA Later
2
Breast Tissue Characterization: Malignant, Benign, and Normal
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Breast tissue specimens (n = 168) were obtained at University College Hospital, Galway. The clinical patient samples comprised of 103 malignant tissue biopsies, 30 normal mammary tissue biopsies obtained at reduction mammoplasty, and 35 fibroadenoma tissues which are benign breast disease tissues. Full patient demographics and clinicopathological details were collected and maintained prospectively (Table 1 ). Samples were immersed in RNAlater® (Qiagen) for 24 hours, then the RNAlater® was removed and the tissue stored at −80°C until required.
| Breast Clinicopathological characteristics | Cancer | Fibroadenoma | Normal |
|---|---|---|---|
| Number of patients | 103 | 35 | 30 |
| Median Patient Age yrs | 56 (35–90) | 44 (17–62) | 46.5 (24–58) |
| Post | 72 | ||
| Pre | 32 | ||
| Invasive Ductal | 78 | ||
| Invasive Lobular | 11 | ||
| Other | 14 | ||
| Luminal A (ER/PR+, HER2/neu-) | 42 | ||
| Luminal B (ER/PR+, HER2/neu+) | 18 | ||
| HER2 Over expressing (ER-, PR-, HER2/neu+) | 16 | ||
| Triple-Negative (ER-, PR-, HER2/neu-) | 16 | ||
| Unknown | 11 | ||
| 1 | 5 | ||
| 2 | 32 | ||
| 3 | 55 | ||
| 1 | 19 | ||
| 2 | 39 | ||
| 3 | 10 | ||
| Stage 1 | 23 | ||
| Stage 2 | 36 | ||
| Stage 3 | 21 | ||
| Stage 4 | 10 |
3
Placental Biopsy Sampling for Gene Expression
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Biopsies were taken from the fetal side of the placenta by cutting into the chorionic plate and sampling of fetal chorionic villi, and preserved in RNALater (Qiagen, USA). To capture variability in gene expression within the placenta, one sampling site was close to the umbilical cord insertion point and one close to the outer margin of the placenta [36 (link)]. We generated gene expression data for 2 biopsies from each of 164 placentas. For some placentas, either tissue was unavailable or the tissue quality and/or quantity were insufficient for analysis, thus, in 12 placentas we relied on 1 biopsy, while we had more than 2 biopsies from 4 placentas. Samples were preserved in RNALater (Qiagen, USA) and stored at -80C. Based on histologic analysis, there was minimal contamination of the biopsies with cells from the chorionic membrane and basal plate [36 (link)].
4
Porcine Tissue Sampling for RNA-seq
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Pigs were slaughtered at a weight of ~100 kg at a commercial slaughterhouse (Danish Crown, Herning, Denmark). Slaughter was performed by submersion into CO2 until unconsciousness ensued followed by exsanguination. Following slaughter, the liver was extracted from the carcass and 150 mg of tissue were retrieved by punch biopsy and immediately immersed into 1.5 ml RNAlater (QIAGEN, Hilden, Germany) in 2 ml Eppendorf tubes (Eppendorf, Hamburg, Germany). The carcasses were kept in a cold room at 4 °C for approximately 1.5 h before 150 mg testis tissue were retrieved by punch biopsy and immersed into 1.5 ml RNAlater (QIAGEN). All samples (n = 96) were stored at −20 °C for 14 days until RNA extraction and sequencing at a commercial facility (AROS A/S, Aarhus, Denmark).
5
RNA Extraction from Tissues and Blood
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On procedure days, 100 μl of blood from K2-EDTA collection tubes was collected prior to centrifugation and was added to 600 μl of AVL viral lysis buffer with 6 μL carrier RNA (Qiagen) for RNA extraction. For tissues, approximately 100 mg was stored in 1 ml RNAlater (Qiagen) for at least 4 days for stabilization. RNAlater was completely removed, and tissues were homogenized in 600 μl RLT buffer and 1% betamercaptoethanol (Qiagen) in a 2 mL cryovial using a tissue lyser (Qiagen) and 0.2 mm ceramic beads. The tissues sampled included axillary and inguinal lymph nodes, liver, spleen, kidney, adrenal gland, lung, pancreas, urinary bladder, ovary or testis, and eye. All blood samples were inactivated in AVL viral lysis buffer, and tissue samples were homogenized and inactivated in RLT buffer prior to removal from the BSL-4 laboratory. Subsequently, RNA was isolated from blood using the QIAamp viral RNA kit (Qiagen), and from tissues using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions supplied with each kit.
6
Tumor and Normal Tissue RNA Extraction
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Fresh samples of tumor and paired normal colonic mucosa were submerged in RNAlater (RNA stabilization reagent, Qiagen, Hilden, Germany) for 24 h at 4 °C and stored at −80 °C after RNAlater removal. Total RNA was extracted using the RNeasy mini kit (Qiagen) and DNase treatment, according to the manufacturer’s procedure, after tissue homogenization with Fastprep-24 (MP Biomedicals, Irvine, CA, USA). RNAs were quantified using a NanodropND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and their quality was assessed using Agilent RNA 6000 Nano kit with a 2100 Bioanalyzer instrument according to the manufacturer’s protocol.
7
Fetal and Placental Tissue Extraction
8
RNA Extraction from Blood and Tissue
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On procedure days, 100 μl of blood from K2-EDTA collection tubes was collected prior to centrifugation, and was added to 600 μl of AVL viral lysis buffer with 6 μL carrier RNA (Qiagen) for RNA extraction. For tissues, approximately 100 mg was stored in 1 ml RNAlater (Qiagen) for at least 4 days for stabilization. RNAlater was completely removed, and tissues were homogenized in 600 μl RLT buffer and 1% Betamercaptoethanol (Qiagen) in a 2 mL cryovial using a tissue lyser (Qiagen) and 0.2mm ceramic beads. The tissues sampled included axillary and inguinal lymph nodes, liver, spleen, kidney, adrenal gland, lung, pancreas, urinary bladder, ovary or testis, and eye. All blood samples were inactivated in AVL viral lysis buffer, and tissue samples were homogenized and inactivated in RLT buffer prior to removal from the BSL-4 laboratory. Subsequently, RNA was isolated from blood using the QIAamp viral RNA kit (Qiagen), and from tissues using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions supplied with each kit.
9
Salmonella Enteritidis Infection in Chickens
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Six male ISA Brown chickens were infected orally at day of hatch with 107 CFU of wild-type Salmonella Enteritidis 147 [15 (link)] or its isogenic SPI1 mutant [16 (link)], and sacrificed 4 days later. Six non-inoculated 5-day-old chickens were included as a control group. Approx. 30 mg of the cecum was collected from each chicken during necropsy, immediately placed into RNAlater (Qiagen) and stored at −80 °C.
In the second experiment, 64 male ISA Brown chickens were infected orally at day of hatch with 107 CFU of wild-type Salmonella Enteritidis 147 and sacrificed on day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 22, 25 and 29 of life, 4 chickens each day. Sixty-eight non-infected chickens were included as controls; four non-infected chickens were sacrificed on day 1 and the remaining at the same time points as the infected ones. During necropsy, approx. 30 mg of the cecum was collected into RNAlater (Qiagen) and stored at −80 °C.
In the second experiment, 64 male ISA Brown chickens were infected orally at day of hatch with 107 CFU of wild-type Salmonella Enteritidis 147 and sacrificed on day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 22, 25 and 29 of life, 4 chickens each day. Sixty-eight non-infected chickens were included as controls; four non-infected chickens were sacrificed on day 1 and the remaining at the same time points as the infected ones. During necropsy, approx. 30 mg of the cecum was collected into RNAlater (Qiagen) and stored at −80 °C.
10
RNA Extraction from Blood and Tissue Samples
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On procedure days, 100 μL of blood from K2-EDTA collection tubes was collected prior to centrifugation and was added to 600 μL of AVL viral lysis buffer with 6 μL carrier RNA (Qiagen) for RNA extraction. For tissues, approximately 100 mg was stored in 1 mL RNAlater (Qiagen) for at least 4 days for stabilization. RNAlater was completely removed, and tissues were homogenized in 600 μL RLT buffer and 1% betamercaptoethanol (Qiagen) in a 2 mL cryovial using a TissueLyser (Qiagen) and 0.2 mm ceramic beads. The tissues sampled included axillary and inguinal lymph nodes, liver, spleen, kidney, adrenal gland, lung, pancreas, urinary bladder, ovary or testis, and eye. All blood samples were inactivated in AVL viral lysis buffer, and tissue samples were homogenized and inactivated in RLT buffer prior to removal from the BSL-4 laboratory. Subsequently, RNA was isolated from blood using the QIAamp viral RNA kit (Qiagen), and from tissues using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions supplied with each kit.
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