PhD Scientific Days 2021

MO_V_L: Molecular Sciences V. Lectures

Lyophilization and homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques

T. Lakat1,2, A. Molnar2, A. Hosszu1,2, B. Szebeni2,3, A. Balogh2, L. Orfi4, A. J. Szabo2,3, A. Fekete1,2, J. Hodrea1,2

1SE Diabetes Research Group, Semmelweis University, Budapest, Hungary
21st Department of Pediatrics, Semmelweis University, Budapest, Hungary
3ELKH-SE Pediatrics and Nephrology Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary
4Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary

Text of the abstract

Introduction:
Lyophilization is a cost-effective method for biological specimen preservation but detailed tissue-specific reference protocols are still lacking. Moreover, data is limited on the long-term stability of proteins and nucleic acids in lyophilized samples. Despite the numerous advantages, freeze drying has not become a routine method in research laboratories yet.
Aims:
We aimed to investigate RNA and protein stability of freeze-dried tissue samples following 20 months of storage at 4°C. We also conducted experiments to investigate the hypothesis that lyophilization and subsequent homogenization can be a superior method for sample preparation when investigating focal organ injuries.
Method:
15 week-old male Wistar rats were sacrificed, kidney, heart, lung, skin and aorta samples were harvested. Samples were cut in two (A, B), the pieces were snap-forzen and A: lyophilized, pulverized and stored at 4°C for 20 months; B: stored at -80°C for 20 months. Western blot (αSMA, pAkt/Akt, pENOS/ENOS) and RT-qPCR (Acta2, Gapdh) measurements were performed to compare the protein and RNA stability, and reproducibility of sample preprocessing in lyophilized and conventional frozen samples. To test reproducibility total protein and RNA were extracted from 4 different parts of frozen samples and 4 times from lyophilized, pulverized homogenates of the same kidney samples.
Results:
Detailed protocols were established for rodent kidney, heart, lung, aorta and skin samples. Mean residual water content of lyophilized samples were below 3.1%. Freeze-drying and storage at 4°C for 20 months does not alter the quality and quantity of examined proteins or RNA molecules that can be extracted from tissue samples. Investigated post-translational modifications are preserved and can be detected even more accurately in lyophilized samples than frozen ones. Western blot and PCR measurements of fibrotic markers from lyophilized, pulverized samples resulted lower scatter and less outlier data points compared to frozen ones.
Conclusion:
Lyophilization proved to be a suitable method for homogenization of biological specimen, which preserves protein and nucleic acid integrity and can improve the reproducibility and reliability of molecular measurements.
Funding: OTKA- PD-131637, FK-124491, K135398, 2017-1.3.1-VKE-2017-00006, 2020-4.1.1.-TKP2020-(6183069269, 6183169273)

University and Doctoral School

Semmelweis University, Károly Rácz Doctoral School of Clinical Medicine