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Travelling with tissue samples in RNAlater

Travelling with tissue samples in RNAlater


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I've been in Brazil collecting some samples (ants) and need to travel back to to the UK with - I've got their brains stored in RNAlater, which have been in the freezer at -4C for a bit less than a month. On the Fisher Scientific website it says that they can be left at 37C for a day in solution before the RNA can begin to degrade. I've got a long journey home which could take up to 3 days in the heat (it's around 38C at the moment here) and involves stopping off through Morocco.

Has anyone travelled with samples in RNAlater like this before in elevated temperatures and got good results from their RNA extractions? Would you reccomend trying to source some dry ice for the journey?


EFFECT OF HOMEMADE RNALATER ON THE QUALITY OF RNA

Extracting intact, high-quality RNA is the first and most critical step in performing molecular biology experiments. High-quality RNA is required for performing the quantitative and qualitative analysis of mRNA expression by RT-PCR, Northern blot hybridization and several nuclease protection assays. RNA isolation from tissue and cell is more challenging. Because these samples rich in endogenous ribonuclease (RNase). It is necessary to minimize the RNase activity to obtain high-quality RNA. So those tissues must be quickly homogenized in a strong protein denaturant usually guanidinium isothiocyanate, or it should be rapidly frozen in liquid nitrogen. Quick freezing and homogenization are even less convenient in the field. So there is a need for the reagent or method that allow preserving the high-quality, intact RNA from tissue sample at ambient temperature. Now a days RNAlater is commercially manufactured by many companies. RNAlater permeates tissues and protects the cellular RNA insitu for 1day at 37°C, one week at 25°C and one month at 4°C and these tissues can also be stored at -20°C for long term usage. However, the cost of commercial RNAlater (C. RNAlater) is higher. So, the protocol for preparing Homemade RNAlater (H. RNAlater) is evolved. The present study was conducted to test the effect of H. RNAlater and C. RNAlater on the quality of the RNA. Moreover, we have also tested the efficiency of Homemade Trizol (H. Trizol) and Commercial Trizol (C.Trizol) on the integrity of RNA.

RNAlater TM RNA Preservation Medium (H. RNAlater) was prepared by following the method of Lader (2001). 200 mg of brain sample from Pangasius hypophthalmus was collected, of which 100 mg was immersed in 1 ml of H. RNAlater reagent and another 100 mg was immersed in 1 ml of C. RNAlater solution and preserved at 4°C. The next day total RNA was isolated from the preserved tissue samples using TRIzol® reagent (Invitrogen, USA) and also by H. Trizol following Sambrook et al. (2001) with slight modifications.

The results indicated that the RNA isolated by using TRIzol® reagent (Invitrogen, USA) from the tissue which preserved in H. RNAlater and C. RNAlater (Invitrogen, USA) gave high quality, intact RNA. But, the integrity of RNA isolated by using H. Trizol was lost. Hence, the result concluded that H. RNAlater is the best alternative to the C. RNAlater.


Yield and Integrity of RNA from Brain Samples are Largely Unaffected by Pre-analytical Procedures

Gene expression studies are reported to be influenced by pre-analytical factors that can compromise RNA yield and integrity, which in turn may confound the experimental findings. Here we investigate the impact of four pre-analytical factors on brain-derived RNA: time-before-collection, tissue specimen size, tissue collection method, and RNA isolation method. We report no significant differences in RNA yield or integrity between 20 mg and 60 mg tissue samples collected in either liquid nitrogen or the RNAlater stabilizing solution. Isolation of RNA employing the TRIzol reagent resulted in a higher yield compared to isolation via the QIAcube kit while the latter resulted in RNA of slightly better integrity. Keeping brain tissue samples at room temperature for up to 160 min prior to collection and isolation of RNA resulted in no significant difference in yield or integrity. Our findings have significant practical and financial consequences for clinical genomic departments and other laboratory settings performing large-scale routine RNA expression analysis of brain samples.

Keywords: Brain tissue Gene expression Pre-analytical factors RNA Integrity Number (RIN) RNA degradation RNA isolation RNA stability.


Travelling with Samples

Following recent issues involving transport dewars being refused as hand or hold luggage at Lyon and Grenoble airports, we would like to remind Users who intend to travel to the ESRF with their sample dewars on the plane that the dewars cannot be taken on-board the plane as hand luggage, and they will only be accepted in the hold as long as the dewars and the cases in which they are contained conform to IATA regulations. Transport cases that do not conform to IATA regulations include mushroom shipping containers. The ESRF will no longer provide airport forms for dewars transported in containers that do not conform to these regulations.

To avoid possible problems with dewars being refused, it is strongly recommended that samples should be shipped by recognised courier companies such as FEDEX, UPS, DHL, World Courier, TNT.

However, if you have to carry samples while travelling, here are some guidelines organised by means of transport. There is a separate section for samples classed as dangerous goods.

Air

Accompanying letters "Airport Forms" can be prepared by the ESRF for Lyon, Grenoble and Geneva airports. These forms were set up in collaboration with the airport authorities. They are used when travelling back to your home institute with samples). These forms can only be used for dry samples liquid samples cannot be carried.

There is a check box for the Airport Form request on the A-form.

Once this box has been checked, an automatically-generated message is sent to the Biosafety Officer who will contact you for extra details. Failing to answer this message will cancel your Airport Form request. Airport Forms prepared in advance by the Safety Group are to be collected from the Experimental Hall Operator office in the Experimental Hall (Ext. 25-25 in front of ID31) after signing various papers.

A few rules are applied for dry shippers transportation:

  • Make sure that you ONLY transport your samples in your dewars
  • Make sure that all liquid nitrogen (forbidden compound on roads, trains and planes) has been poured off before shipping your dry shippers
  • Make sure that your samples, dewars and the cases in which they are contained comply with the IATA regulations.

Train

For transportation of your samples using the Eurostar, the ESRF does not provide a form for such travel. However, you are invited to contact the French Customs office in Paris, whose details are below, in order to obtain clearance (a specific letter) for travelling with your samples.

Users must send a letter to the following address:

Pôle Action Economique

Direction Régionale des Douanes de Paris

16, rue Yves Toudic

75010 PARIS

email address : [email protected]

The following information should be provided in this letter:

  • Type of samples concerned
  • The reason for their transport
  • The name(s) of the person(s) responsible for the samples during their transport
  • The date and time of the train on which the samples will be carried.

On receipt of this letter a special dispensation will be sent to the Users concerned, and a copy to the Safety Control Brigade, so that their containers will not be opened on the Eurostar site. However, containers will still need to pass the X-ray control.

Scientists may also contact the Eurostar Security Standards Management Security Pass office at St Pancras International, Pancras Road, London, NW1 2QP.

Road

Transportation of samples by road must comply with the ADR regulations, specifying several classes of dangers depending on sample types. Original text is available via this link

Transporting dangerous goods

For dangerous goods or compounds, there is a specific regulation that applies, one for each type of transport.

Transport Mode

Applicable Texts

Decree 1 July 2001 modified 9 May 2008 relating to the transport of dangerous goods by road (ADR regulations).

Regulation concerning dangerous materials using the international rail transport of dangerous goods (RID regulations).

Rules of the International Air Transport Association (IATA) and the International Civil Aviation Organisation (ICAO) for technical instructions.

European agreement relating to the international transport of dangerous materials and goods by internal navigation traffic means (ADNR regulations).

International Maritime Dangerous Goods code (Code IMDG).

Transported dangerous goods are referenced according to 9 classes, depending on their potential risk:

Explosive materials and objects

Compressed gases, liquefied or dissolved under pressure

4.1 : Flammable solids,
4.2 : Substances liable to spontaneous combustion,
4.3 : Substances that on contact with water emit flammable gases


CDNA-arrays and real-time quantitative PCR techniques in the investigation of chronic achilles tendinosis

The aetiology and pathogenesis of chronic painful Achilles tendinosis are unknown. This investigation aimed to use cDNA arrays and real-time quantitative polymerase chain reaction (real-time PCR) technique to study tendinosis and control tissue samples. Five patients (females mean age 57.1 ± 4.3 (years ±SD)) with chronic painful Achilles tendinosis were included. From all patients, one biopsy was taken from the area with tendinosis and one from a clinically normal area (control) of the tendon. The tissue samples were immediately immersed in RNAlater and frozen at –80°C until RNA extraction. Portions of pooled RNA from control and tendinosis sites, respectively, were transcribed to cDNA, radioactively labelled ( 32 P), hybridized to cDNA expression arrays, and exposed to phosphoimager screens over night. Expressions of specific genes, shown to be regulated in the cDNA array analysis, were analyzed in the individual samples using real-time PCR. cDNA arrays showed that gene expressions for matrix-metalloproteinase-2 (MMP-2), fibronectin subunit B (FNRB), vascular endothelial growth factor (VEGF), and mitogen-activated protein kinase p38 (MAPKp38) were up-regulated, while matrix-metalloproteinase-3 (MMP-3) and decorin were down-regulated, in tendinosis tissue compared with control tissue. Using real-time PCR, ⅘ and ⅗ patients showed up-regulation of MMP-2 and FNRB mRNA, respectively. For decorin, VEGF, and MAPKp38, real-time PCR revealed a great variability among patients. Interestingly, the mRNAs for several cytokines and cytokine receptors were not regulated, indicating the absence of an inflammatory process in chronic painful Achilles tendinosis. In conclusion, cDNA-arrays and real-time PCR can be used to study differences in gene expression levels between tendinosis and control tendon tissue. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.


Technical Validation

RNA sequencing data quality control and consistency tests

To assess if the obtained gene expression profiles are in correlation with the biological nature of the biosamples tested and identify samples that might be of low quality, we performed cluster analysis of all the RNA sequencing data we had obtained (Fig. 1). The color on the dendrogram indicates technical type of biosamples: FFPE or tissue in RNAlater for solid tissue, and RNA in ethanol for blood samples.

The hierarchical clustering dendrogram of all experimental RNA sequencing profiles of human tissues. Gene expression data were used to calculate Euclidian distances between the samples. Color indicates the sample preparation method (tissue in FFPE, RNA in ethanol, tissue in RNAlater). The lower scale indicates the number of uniquely mapped reads. QC denotes the quality control threshold of 2.5 million uniquely mapped reads.

The FFPE and RNAlater samples formed several mixed clusters on the dendrogram, which suggests that the clustering was independent of the sample preparation technique for solid tissues. The blood samples formed a clearly distinct cluster on the left side of the dendrogram, which is in line with the histological status of this tissue (Fig. 1), which stand apart among other samples.

However, we then observed a mixed cluster next to the one formed by the blood samples, which included both blood and solid tissue specimens (Fig. 1, red), and such clustering did not correspond to physiological origin of biosamples. We noticed that that cluster was formed exclusively by the samples with relatively low (less than 2.5 million) number of uniquely mapped sequencing reads (Fig. 1, scale) and hypothesized that this may represent a deviation which arose due to insufficiency of data. We analyzed contents of the samples with respect to the number of uniquely mapped reads (Fig. 2) and found that the samples with lowest number of reads indeed formed a distinct cluster with the upper threshold of

2.5 million reads uniquely mapped to HGNC genes (Fig. 2). We, therefore, used this threshold, which enabled us to separate effectively solid tumors from hematological ones, as an indicator for quality control (QC) of the sequenced gene expression profiles. 132 samples out of a total of 159 passed the QC threshold (Table 1). After the QC filter was applied RNA sequencing profiles became clustered in the hierarchical way following the histological origin of the tissues (Fig. 3).

The distribution of the experimental RNA sequencing profiles with respect to the number of uniquely mapped reads. The vertical dashed line indicates the QC threshold of 2.5 million uniquely mapped reads per sample.


Difficulty in cutting tissue after collection of sample in RNAlater - (Sep/14/2012 )

I kept my tissue samples in RNAlater after collection from the OT.
The samples were kept overnight in 4 degrees, and transferred to -80 degrees the next day.

When trying to cut the tissue samples with cryostat,
the samples seem to split and break and I cannot prepare my slides with this samples.
Before subjecting my samples to the OCT medium, I just dab the samples on a tissue paper to get rid of the RNAlater.

So do the difficulty in cutting the tissue samples are caused by RNAlater ?
How to remove the RNAlater covering the tissue sample effectively before subjecting them for slide preparation ?

Hope that someone out there can help me to overcome this problem.

RNAlater is meant to save RNA from degradation. It is not mean to keep the tissue at its original shape. We always cut our sample, save some of it in RNAlater, and keep the rest in other buffers.

Yeah, RNAlater is mostly a sulphate, which is used to precipitate proteins, not preserve them. If you want to preserve tissue properly, use a fixative such as formaldehyde.

Thanks guys for your comments.

I have just came across an article that mentioned to incubate the tissue collected in RNAlater in ice-cold PBS for 5-10mins before cutting them using cryostat.


Protein isolation from RNAlater stored tissue - (Jul/08/2008 )

Has anyone done western and IP using protein isolated from tissue stored for a brief time (1 hour) in RNAlater (ambion)?

If not, do you see any theoretical problem with it?

Alright. As I generally do, I called up tech support and Yes, you can definitely do the Western.

http://www.ambion.com/techlib/tn/113/6.html

IP is not so sure, but there is at least one publication to it.

Problem solved. Still, any dreadful stories of personal experience are most welcome

Has anyone done western and IP using protein isolated from tissue stored for a brief time (1 hour) in RNAlater (ambion)?

If not, do you see any theoretical problem with it?

Out of curiosity I googled RNAlater and this is what I found:

Isolating protein from RNAlater Solution-stored samples
Proteins are also preserved in RNAlater Solution. RNAlater
Solution will denature proteins therefore, protein obtained
from samples stored in it will be suitable for applications such
as Western blotting or 2D gel electrophoresis, but not for
applications that require native protein.

So, I would probably rule it out for IP if I were you.

Has anyone done western and IP using protein isolated from tissue stored for a brief time (1 hour) in RNAlater (ambion)?

If not, do you see any theoretical problem with it?

Out of curiosity I googled RNAlater and this is what I found:

Isolating protein from RNAlater Solution-stored samples
Proteins are also preserved in RNAlater Solution. RNAlater
Solution will denature proteins therefore, protein obtained
from samples stored in it will be suitable for applications such
as Western blotting or 2D gel electrophoresis, but not for
applications that require native protein.

So, I would probably rule it out for IP if I were you.

Thanks smu. In fact the pubmed ID article they gave is about IHC, not IP, so it makes sense.

However, I will try out IP too, it all depends upon whether my Ab will recognize the denatured protein. As long as it does not crosslink proteins (which it doesn't), I can always try. Will let you know in this thread. Give me a week

Has anyone done western and IP using protein isolated from tissue stored for a brief time (1 hour) in RNAlater (ambion)?

If not, do you see any theoretical problem with it?

Out of curiosity I googled RNAlater and this is what I found:

Isolating protein from RNAlater Solution-stored samples
Proteins are also preserved in RNAlater Solution. RNAlater
Solution will denature proteins therefore, protein obtained
from samples stored in it will be suitable for applications such
as Western blotting or 2D gel electrophoresis, but not for
applications that require native protein.

So, I would probably rule it out for IP if I were you.

Thanks smu. In fact the pubmed ID article they gave is about IHC, not IP, so it makes sense.

However, I will try out IP too, it all depends upon whether my Ab will recognize the denatured protein. As long as it does not crosslink proteins (which it doesn't), I can always try. Will let you know in this thread. Give me a week


Optimization of preservation and storage time of sponge tissues to obtain quality mRNA for next-generation sequencing

Transcriptome sequencing with next-generation sequencing technologies has the potential for addressing many long-standing questions about the biology of sponges. Transcriptome sequence quality depends on good cDNA libraries, which requires high-quality mRNA. Standard protocols for preserving and isolating mRNA often require optimization for unusual tissue types. Our aim was assessing the efficiency of two preservation modes, (i) flash freezing with liquid nitrogen (LN₂) and (ii) immersion in RNAlater, for the recovery of high-quality mRNA from sponge tissues. We also tested whether the long-term storage of samples at -80 °C affects the quantity and quality of mRNA. We extracted mRNA from nine sponge species and analysed the quantity and quality (A260/230 and A260/280 ratios) of mRNA according to preservation method, storage time, and taxonomy. The quantity and quality of mRNA depended significantly on the preservation method used (LN₂) outperforming RNAlater), the sponge species, and the interaction between them. When the preservation was analysed in combination with either storage time or species, the quantity and A260/230 ratio were both significantly higher for LN₂-preserved samples. Interestingly, individual comparisons for each preservation method over time indicated that both methods performed equally efficiently during the first month, but RNAlater lost efficiency in storage times longer than 2 months compared with flash-frozen samples. In summary, we find that for long-term preservation of samples, flash freezing is the preferred method. If LN₂ is not available, RNAlater can be used, but mRNA extraction during the first month of storage is advised.


Expression of BMI-1 and Mel-18 in breast tissue--a diagnostic marker in patients with breast cancer

Background: Polycomb Group (PcG) proteins are epigenetic silencers involved in maintaining cellular identity, and their deregulation can result in cancer. Expression of Mel-18 and Bmi-1 has been studied in tumor tissue, but not in adjacent non-cancerous breast epithelium. Our study compares the expression of the two genes in normal breast epithelium of cancer patients and relates it to the level of expression in the corresponding tumors as well as in breast epithelium of healthy women.

Methods: A total of 79 tumors, of which 71 malignant tumors of the breast, 6 fibroadenomas, and 2 DCIS were studied and compared to the reduction mammoplastic specimens of 11 healthy women. In addition there was available adjacent cancer free tissue for 23 of the malignant tumors. The tissue samples were stored in RNAlater, RNA was isolated to create expression microarray profile. These two genes were then studied more closely first on mRNA transcription level by microarrays (Agilent 44 K) and quantitative RT-PCR (TaqMan) and then on protein expression level using immunohistochemistry.

Results: Bmi-1 mRNA is significantly up-regulated in adjacent normal breast tissue in breast cancer patients compared to normal breast tissue from noncancerous patients. Conversely, mRNA transcription level of Mel-18 is lower in normal breast from patients operated for breast cancer compared to breast tissue from mammoplasty. When protein expression of these two genes was evaluated, we observed that most of the epithelial cells were positive for Bmi-1 in both groups of tissue samples, although the expression intensity was stronger in normal tissue from cancer patients compared to mammoplasty tissue samples. Protein expression of Mel-18 showed inversely stronger intensity in tissue samples from mammoplasty compared to normal breast tissue from patients operated for breast cancer.

Conclusion: Bmi-1 mRNA level is consistently increased and Mel-18 mRNA level is consistently decreased in adjacent normal breast tissue of cancer patients as compared to normal breast tissue in women having had reduction mammoplasties. Bmi-1/Mel-18 ratio can be potentially used as a tool for stratifying women at risk of developing malignancy.


Watch the video: How to isolate RNA from tissue or cells (December 2022).