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Advancement in Preserving Human Tissue Morphology and Gene Expression Profiling

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The quality of your results is directly impacted by the quality of the human samples procured and whether the tissues have retained the morphology and integrity of their RNA. Current preservation techniques range from stopping all processes (formalin), to suspending all processes (feeezing), to targeting specific processes (RNAlater®).

A significant advance would be the ability to keep the tissues alive. Previous attempts to preserve the integrity of RNA in human tissue biopsies have focused on using non-fixed, frozen tissues in order to overcome the fragmentation of RNA. However, the logistics of preparing frozen tissue samples is compromised by having to process the ex vivo tissue samples almost immediately (1-3 hours).

Micke et al., (2006) produced high quality RNA profiles from fresh, frozen human tissue biopsies that were suitable for gene expression. The authors showed that tissue samples stored, immersed in ‘cold’ 0.9% sodium chloride solution, in comparison to storage in RNAlater® solution (Ambion, USA), exhibited less tissue shrinkage and, more significantly, minimal changes in gene expression patterns following incubation periods of up to 16 hours, the best results being obtained up to 6 hours after biopsy.

Other implications of this work are to suggest that time is limited and that RNA later® may impact upon tissue viability. The value of gene expression profiling relates to an understanding of the pathogenetic mechanisms which govern diagnostic and prognostic outcomes. It is an essential requisite in the preservation of tissue that any aberrant changes in gene expression profiling or replication be negated and that RNA integrity is maintained and mRNA is at representative levels.

Trial A: AQIX® RS-I compared to RPMI

Aims. To compare the preservation of human colon tissue biopsies stored and transported over ice for a period of 30 hours using RPMI culture media verses samples immersed in either AQIX® RS-I or RS-S solutions. All samples were transported using AQIX® Storage & Transportation Kits (Code: RSI/KIT).

Method. Human Colon Tissue Biopsies: Biopsied samples (1cm x 1cm) of normal human colon tissue were procured and placed directly into Nalgene® bottles containing either 125 ml of AQIX® RS-I, AQIX® RS-S or RPMI solutions (see SOP/2009, below) and transported over ice for a period of 30 hours.

Results. Human Colon Tissue Biopsies: Tissue samples stored and transported in AQIX® RS-I solution showed superior preservation of both morphology and RNA integrity in comparison to those tissue samples stored and transported in RPMI solution

Fig. 2    RNA QC of Normal Human Colon Biopsies Transported and Stored in RPMI and AQIX® RS-I media

•30h post operation

•Transportation on ice

Histological examination of tissue samples was carried out using conventional H & E techniques in association with an evaluation of RNA integrity (e.g., RNA concentration; rRNA ratio;RIN).

Trial B: AQIX® RS-I tissue preservation compared over time

 Aims.         To evaluate the preservation of normal and cancerous  human breast tissue biopsies stored and transported over ice for periods of 24, 36 and 48 hours using AQIX® RS-I solution and contained in AQIX® Storage & Transportation Kits (Code: RSI/KIT).

 Method.    Normal and Cancerous Human Breast Tissue  Biopsies: Biopsied samples (approximately1cm x 1cm) of normal and cancerous human breast tissue were procured and placed directly into Nalgene® bottles containing 125 ml of AQIX® RS-I solution (see SOP/2009, below) and transported over ice for periods of 24, 36 and 48 hours.

 Results. Normal and Cancerous Human Breast Tissue Biopsies: Tissue samples stored and transported in AQIX® RS-I solution showed excellent preservation of both morphology and RNA integrity over 24, 36 and 48 hours. Noteably, the ratio of 28S:18S ribosomal subunits (rRNA) remained high in all three breast cancerous tissue samples at 24 hours and the majority of samples at 36 hours. RNA integrity (RIN) was better preserved in the majority of the breast cancerous tissue samples over the 24-48 hours in comparison to normal breast tissue samples.

 

Fig.3       Fresh breast cancer tissue morphology and RNA integrity following 24 – 48h of transportation in AQIX® RS-I solution

 

 

  

Discussion

 Acknowledging that RNA is considered a most fragile molecule along with the instability of the cytoarchitecture of malignant or cancerous cells upon isolation (Ohashi et al., 2004), both the morphology and RNA composition of the human breast tissue samples in this study show remarkable preservation over the 24 – 48 hour period of storage and transportation over ice in AQIX® RS-I solution. This is a significant observation compared to previously published procurement techniques.

 In summary, the application of AQIX® fluid technology in this study has shown superior preservation of tissue morphology and superior RNA integrity (RIN’s) in comparison to other commercially available products.

 Conclusion

 The results presented indicate that AQIX® RS-I solution significantly advances the ability to store and transport cancerous tissue samples over periods of time in a condition that will preserve their morphology, RNA integrity and thereby facilitate more accurate expression of gene profiles to advance diagnostic and prognostic outcom

References:

1. Patrick Micke, Mitsuhiro Ohshima, Simin Tahmasebpoor, Zhi-Ping Ren, Arne Östman, Fredrik Pontén and Johan Botling. Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens. Laboratory Investigation (2006);86:202; 86:202– 211

2. Ohashi, Y., Creek, K.E., Pirisi, L., et al. RNA degradation in human breast tissue after surgical removal: a time- course study. Exp Mol Pathol 2004;77:98-103.

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