gastric, biliary, oesophageal and bronchial brushings, bronchial washings/ lavages and endobronchial ultrasound bronchoscopy (EBUS)) should be sent in CytoRich red fluid. All subjects signed the informed consent. The minimum volume allows one single analysis including instrument dead volume. 7 . Light As shown in Fig 1A, the 260/280 ratio remained consistent in different sets, demonstrating the high purity of RNA samples. Therefore, the less degraded the RNA samples are, the more consistent Ct values the RNA samples have. The RNU6B expression was quantified using TaqMan® Fast Universal PCR Master Mix (2✕), No AmpErase® UNG (Applied Biosystems) and StepOnePlus TM System. As a result, 210 blood tubes were collected (3 replications * 7 conditions * 10 subjects). When the samples were stored longer than 24 hours, the Ct values of U6 increased up to approximately 27 with little variations. Deliver to the laboratory as soon as possible after collection and follow temperature and storage guidelines indicated for … So far, our result showed that, with long storage duration, the quality of DNA did not decline but the concentration of DNA did. (c) The methylation intensities in Fig 4B were normalized based on the corresponding loading controls and plotted. The RT master mix was prepared as follow: 0.3 μl 100Mm dNTPs, 3 μl reverse transcriptase, 1.5 μl reverse transcription buffer, 0.19 μl RNase inhibitor, 6 μl primer, 1.01 μl nuclease-free water and 3 μl RNA. For more information click here. Conceptualization, 514 0 obj <>/Filter/FlateDecode/ID[<67B40D6B09F1BA418490B4DC0190E5A6><1ECB3226651F344D97179C2A0D183DAB>]/Index[495 45]/Info 494 0 R/Length 92/Prev 51046/Root 496 0 R/Size 540/Type/XRef/W[1 2 1]>>stream So, the storage duration and storage temperature, from blood collection to nucleic acid extraction, are critical to high-quality DNA and RNA samples. Funding: This article was funded by the grants from the Ministry of Science and Technology, Taiwan (MOST 106-2311-B-182A-002) and from Chang Gung Memorial Hospital (CMRPG8F0591 and CLRPG8D0113). Gently invert the tube eight (8) times immediately after collection to evenly distribute the additive. Fig 2E showed that higher expression abundance of IP-10 is expected in longer storage durations. In animal, lymphocytes in circulation system function as protectors, conducting many immune activities to protect animals from external damage, chemical or biological. Watch videos. In the absence of protocols or recommendations from manufacturers, then validated protocols for sample storage and preparation must be defined. The conclusion of Fig 4A was acquired by examining only one specific CpG marker on only one subject. No. Although with larger variations than 18S and GAPDH, the expression of IP-10 is activated by stress in spite of RNA degradation. Blood was collected using scalp vein set (NIPRO, Osaka, Japan), vacutainer® One-Use holder and Vacutainer® Blood Collection Tubes (with heparin, REF367874, BD, New Jersey, USA). The Ct values of U6 and 18S significantly rose at the duration of 32hr. For information on the collection of seminal fluid samples for fertility analysis: Click Here, For information on the collection of seminal fluid for post vasectomy analysis: Click Here. In spite of the contradiction, our finding suggested that the blood samples should be stored for no longer than 24 hours at room temperature to obtain the best quality of RNA samples.

S2 Fig showed that the DNA samples with different durations aggregated with the size larger than 10K base pair. Shipping . Yes Read articles. 229 0 obj <>stream No, Is the Subject Area "RNA extraction" applicable to this article? For more information about PLOS Subject Areas, click 61255, Active Motif) at 4°C overnight. DNA samples (100ug) were added to denaturation buffer (0.4mM NaOH and 10mM EDTA) and denatured at 100°C for 10min. Specimen bags should be clearly marked to indicate the temperature at which the specimen needs to be maintained. Therefore, in addition to RNA quality examination, we also investigated the expression abundances on three common internal control genes with qPCR assay. Exposing a specimen to light causes a breakdown of some analytes such as bilirubin. These digits are RIN value, 260/280 value, qPCR Ct value, WBC count, WBC concentration or methylation percentage. endstream endobj 496 0 obj <. If samples are collected after the transport has left then they should be placed in a refrigerator  except those samples for genetic tests (EDTA), HLA B27 (EDTA), joint aspirates or other “sterile fluids”, urethral swabs and HVS. Blood Sciences Samples: Minimising Deterioration Whole Blood Fig 2A brought the conclusion, similar with Fig 1A, that 24hr is a golden duration for high-quality RNA sample. In summary, storage of whole blood in low temperature is not recommended. * denoted significance by t-test. Next, the isolated RNA samples were eluted with 50 μl elution buffer. Yes �� 9��Pi�Å�R�*::*:�;:*:�

No, Is the Subject Area "RNA sequencing" applicable to this article? So, the more WBCs lysed, the less DNA concentration. The correctness of physical test and the success of biomedical study largely depend on high-quality DNA and RNA samples from blood. CSF, pleural fluid, joint fluids and pus require culturing without delay. Investigation, No, Is the Subject Area "Blood cells" applicable to this article? Compared with fresh condition (D0), cg08298591 was differentially methylated at the duration point D7 (P<0.05). From the preliminary result, 32hr is a clear duration point at which RIN value started to decline. The real-time PCR master mix was prepared as follow: 7.5 μl 2X master mix, 0.8 μl probe, 6.7 μl nuclease-free water and 5 μl cDNA. Definition of Human Biospecimens These guidelines apply to all human biospecimens , including--but are not limited to--blood So, storage duration DID affect DNA methylation at consistent temperature. Minimum acceptable sample is 20 grams. Yes

Previous studies tried to develop the protocol for dry DNA storage and shipment [22]. Carefully follow the instructions in the kit. P 7W}���]� |_��'��U�Y. For the ≦24 hr samples, although the Ct values of 18S varied, most of them were less than 10 (mean = 9.61, stand deviation = 0.96). The samples in FR set were stored at freezer immediately after blood collection. The raw data for plotting each figure and sub–figure is available in S1 Table. Fixed samples do not need to go in the fridge, but refrigeration will not cause any deterioration. Samples may be ethylenediamine tetra-acetic acid (EDTA) or clotted. Overnight storage of blood samples before dispatch to the laboratory is not recommended and actively discouraged. Blood from humans can infect people with diseases that the donors carried. Samples may be ethylenediamine tetra-acetic acid (EDTA) or clotted. These Guidelines recognise that blood can be a hazard. In this study, statistical analyses were conducted with Microsoft Excel. For more information about PLOS Subject Areas, click DNA samples from WBC are also usually used for evaluating DNA methylation [19, 20].

Compared with room temperature, low temperature did not protect genes from further degradation. PLOS ONE promises fair, rigorous peer review, Gently invert the tube eight (8) times immediately after collection to evenly distribute the additive. As shown in Fig 3C, at room and low temperature, longer storage duration also caused lower DNA concentrations. As shown in this Fig, the samples with high RIN value have relatively strong and clear 18S and 28S peaks. Therefore, examining the concentration of these chemicals in blood may indicate one's physical conditions, especially for metabolic conditions. %PDF-1.6 %���� We also observed that NEUT-X, a routine parameter useful in detecting myelodysplastic syndrome, became unreliable when analyzed 24 h after sample collection. With longer duration, no matter at room or low temperature, global methylation intensities decreased. Although the fresher the better is the golden rule when dealing with clinical samples, including liquid biopsies, we are interested in how much storage duration and storage temperature of blood affect the qualities of DNA and RNA. All blood samples were collected from one single subject and the storage temperature is 24°C.