After a 15-wk period of GAA supplementation, hen and progeny tissues from the 0.15% GAA and control groups were collected for mRNA relative expression analyses as follows: 6 fertile eggs from each group (one per hen) were collected from each group and incubated in a Petersime hatchery (Zulte, Belgium) under standard conditions (37.5°C, 60% RH). Subsequently, 6 hens from each group were sacrificed by cervical dislocation. The following tissues were immediately collected and stored in RNA save (Biological Industries, Beit Haemek, Israel): the duodenum, jejunum and ileum segments of the small intestine and the ovary, magnum and isthmus of the reproductive tract. At the final stage of egg incubation, progeny (embryos and hatchlings) were killed by cervical dislocation at embryonic ages E17, E19, and day of hatch (DOH ). Their YST, small intestines, kidneys, and livers were removed and stored in RNA save (Biological Industries, Beit Haemek, Israel). Total RNA was isolated from all tissue samples using Tri-Reagent (Bio-Lab, Jerusalem, Israel) as per the manufacturer's protocol. cDNA was synthesized from 1.0 μg total RNA using the RevertAid RT-PCR Kit (Thermo Fisher Scientific, Tamar, Mevaseret-Zion, Israel) in a T100 Bio-Rad instrument (Hercules, CA, USA), as per the manufacturer's protocol.
Rna save
Manufactured by Sartorius
Sourced in Israel, Canada
RNA Save is a stabilization reagent designed to protect RNA samples from degradation during storage and transportation. It preserves the integrity of RNA molecules, allowing for accurate and reliable analysis.
Automatically generated - may contain errors
Lab products found in correlation
Neutral buffered formalin, by Avantor (4 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Optimum cutting medium oct, by Leica (2 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Unitranz rt transport system, by Puritan (1 mentions)
Oct compound, by Avantor (1 mentions)
Epitect control dna, by Qiagen (1 mentions)
Hpaii, by New England Biolabs (1 mentions)
Dulbecco s pbs, by Merck Group (1 mentions)
20 protocols using rna save
1
Broiler Chicken GAA Supplementation Effects
2
RNA Extraction and qPCR Analysis of Bladder Tissue
3
RNA Extraction and qPCR Analysis of Bladder Tissue
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Bladders were flash frozen or stabilized in RNA Save (01–891-1A, Biological Industries) and RNA extracted using TRIzol reagent (15596018, Invitrogen) according to manufacturer protocol followed by gDNA digestion with TURBO DNA-free kit (AM1907, Invitrogen). cDNA was generated using Superscript II Reverse Transcriptase (18064014, Invitrogen). qPCR was performed with SsoAdvanced Universal SYBR Green Supermix (1725275, Bio-Rad) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Fold-changes were calculated using Ct method and normalized internally to 18S expression.
4
Euthanasia and Necropsy Protocol
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Following euthanasia, necropsy was conducted to detect the gross lesions, and the length of the oviducts was measured. The oviduct washes were collected by injecting and gently massaging 10 ml of cold PBS along the entire length of the oviduct. Samples were taken from the trachea, lung, kidney, cecal tonsils, ovary, magnum, isthmus, and uterus and preserved in RNA Save® (Biological Industries, Beit Haemek, Israel) for storage at −80 °C. Additionally, the samples were also stored in 10% neutral buffered formalin (VWR International, Edmonton, AB, Canada).
5
Tissue Sampling and Blood Collection Protocol for Avian Study
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At 7 dpi, under isoflurane anesthesia, 1 mL of intracardiac blood was collected from four chicks of the infected group and five chicks of the control group followed by euthanasia through cervical dislocation. Portions of trachea, lung, kidney, ovary, oviduct, and cecal tonsils (CT) were collected in RNA Save® (Biological Industries, Beit Haemek, Israel) and Optimum Cutting Temperature (OCT) compound (VWR International, Edmonton, AB, Canada). Additionally, portions of trachea, lung, and kidney were collected in 10% neutral buffered formalin (VWR International, Edmonton, AB, Canada). At 14, 21, 28, 35, and 91 dpi, l ml of wing vein blood was collected from all birds in both groups.
At 112 dpi (16 weeks of age), all the remaining birds in both groups were euthanized by cervical dislocation under isoflurane anesthesia. Portions of kidney, ovary, oviduct, and cecal tonsils were collected in RNA Save. In addition, portions of previously mentioned tissues, except cecal tonsil, were fixed in 10% neutral buffered formalin.
Tissues collected in RNA Save and OCT compound were preserved at −80 °C until processing. The gross anatomy of the birds that died or were euthanatized during the experiment was examined and recorded.
At 112 dpi (16 weeks of age), all the remaining birds in both groups were euthanized by cervical dislocation under isoflurane anesthesia. Portions of kidney, ovary, oviduct, and cecal tonsils were collected in RNA Save. In addition, portions of previously mentioned tissues, except cecal tonsil, were fixed in 10% neutral buffered formalin.
Tissues collected in RNA Save and OCT compound were preserved at −80 °C until processing. The gross anatomy of the birds that died or were euthanatized during the experiment was examined and recorded.
6
PBMC Isolation from Whole Blood
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PBMCs were separated from whole blood with a density gradient tube (Uni-Sep, Novamed) as follows: 10 to 15mL EDTA treated blood were added to the density gradient tube, followed by centrifugation at1000g for 30 minutes. PBMC layer was then carefully removed. Cells were washed twice with PBS and 200µl of RNA save (Biological Industries, Beit Haemek, Israel) were added. Samples were kept overnight at 4°C and stored at –80°C.
7
Placental Specimen Collection Protocol
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After collection of cord blood, the umbilical cord and membranes are removed from the placenta; the blood is drained; and the placenta is weighed to the nearest 0.5 g (iBALANCE i2500; My Weigh Canada, Vancouver, BC, Canada). Placental specimens from at least two quadrants of the placenta are collected and divided into three specimen types: (1) full disc biopsies of approximately 0.5-cm thickness that are stored in 10 % formalin at room temperature for histopathological examination; (2) small tissue samples (approximately 0.2 cm3) from the fetal surface of the placenta that are stored in RNA Save (Biological Industries Israel Beit Haemek, Kibbutz Beit Haemek, Israel) at 2–8 °C for 48 h and then transferred to −80 °C until epigenetic and/or gene expression studies are done; and (3) small tissue samples (about 0.2 cm3) from the maternal surface of the placenta that are stored and used similarly to the specimens from the fetal surface. Specimens from each quadrant are collected and pooled to create aliquots that represent the entire placental disc (one pooled histopathology sample and multiple pooled RNA Save samples from each of the fetal and maternal sides).
8
Maxillary Periodontal Tissue Harvesting
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Periodontal tissue homogenate was collected as previously described39 (link). In brief, maxillae were harvested and trimmed immediately after sacrifice. All processes were performed on ice. The soft tissues were discarded. The maxillary samples ranged antero-posteriorly from the mesial part of the first molar to the distal margin of the third molar, and latero-laterally from the buccal outer margin of the molars to the palatal axis close to the molars. Bone depth was ~ 1 mm, depending on alveolar bone thickness. Samples were kept in RNAsave (Biological Industries, Beit Ha’emek, Israel), snap-frozen in liquid nitrogen and stored at −80 °C until RNA extraction.
9
Evaluating Mn-CDs-NHF Treatment in 4T1 Breast Tumor
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4T1 mouse breast carcinoma cells were suspended in RPMI medium/Matrigel (1:1) (1 × 106 in 50 μL) and injected into the mammary pad of female BALB/c mice under depth anesthesia, as previously described [15 (link)]. At two weeks post tumor cells inoculation, mice had been started to be treated via intraperitoneal injection (twice per week) with 10% (100 µg/mL) Mn-CDs-NHF (n = 6) or Gadovist (n = 6) for 3 weeks. CD-NHF concentration is relative to mice blood volume and represents one of the previously tested concentrations [15 (link)].
At the end of the testing period, the animals were euthanized (neck dislocation under deep anesthesia), and primary tumors and various organs (liver, kidneys, lungs, spleen) were collected. Half of each primary tumor and half of each lung were immediately suspended in RNA Save (Biological Industries, New Haven, CT, USA) and stored at −80 °C until use. The other half from each primary tumor and each lung were preserved in 10% paraformaldehyde (Sigma-Aldrich) for further analysis.
At the end of the testing period, the animals were euthanized (neck dislocation under deep anesthesia), and primary tumors and various organs (liver, kidneys, lungs, spleen) were collected. Half of each primary tumor and half of each lung were immediately suspended in RNA Save (Biological Industries, New Haven, CT, USA) and stored at −80 °C until use. The other half from each primary tumor and each lung were preserved in 10% paraformaldehyde (Sigma-Aldrich) for further analysis.
10
Chicken IBV Isolates Infection Study
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Two groups of 6 day-old unsexed-SPF chickens (n = 7 per group) were infected with 50 μL inoculum containing 2.75× 104 PFUs of IBV isolates 15AB-01 and 15SK-02 intra-tracheally under isoflurane anesthesia. Another 5 of 6-day old SPF chickens were kept as uninfected controls. The chickens were weighed at 0, 2, 3, 4, 10, 12 and 14 dpi. At 2, 3, 10 and 12 dpi, oropharyngeal and cloacal swabs were obtained using Puritan® Unitranz-RT transport system (Puritan Medical Products LLC, Maine, USA) and stored at − 80 °C until further processing. At 4 dpi, 4 chickens from each infected and 3 chickens from the uninfected group were euthanized and trachea, lung, kidneys, cecal tonsils and reproductive tract samples were collected into RNASave® (Biological Industries, Beit Haemek, Israel) and stored at -20 °C. In addition, a portion of trachea, lung, kidneys, cecal tonsils and reproductive tract samples were collected into 10% neutral buffered formalin for histopathological examination and in Optimum Cutting Medium (OCT, Leica Biosystems, Wetzlar, Germany) for immunostaining. The remainder of chickens were continued to be observed and tissue samples were collected in RNASave® for genome load quantification at 14 dpi.
For the sampling of tissues, the chickens were euthanized using overdose of isoflurane anesthesia followed by cervical dislocation.
For the sampling of tissues, the chickens were euthanized using overdose of isoflurane anesthesia followed by cervical dislocation.
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