Spatially mapped gene expression analysis from tissue

Spatial gene expression analysis;RT-LAMP;lab-on-a-chip;microwell array

Ganguli, Anurup ; Bashir ; Rashid

关键词: Spatial gene expression analysis;    RT-LAMP;    lab-on-a-chip;    microwell array;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/90974/GANGULI-THESIS-2016.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Spatial gene expression analysis platforms have been widely used for a variety of applications ranging from pathogen detection to the analysis of spatial variation of an mRNA in a tissue section. Traditionally, this is accomplished by using in situ polymerase chain reaction (in situ PCR) or in situ hybridization (ISH). In addition, laser capture microdissection (LCM) followed by RT-qPCR of the locally captured tissue has also been used to study these spatial variations at a molecular level. But, all of the above techniques are plagued with different issues such as low sensitivity in the case of ISH, low reproducibility and long experimental run time for in situ PCR, and long sample acquisition and purification times for the LCM based techniques. This calls for a method that can reliably, rapidly and with high sensitivity perform spatial gene expression analysis starting from a tissue sample. We present a novel approach that combine microfabrication techniques with reverse transcription-loop mediated isothermal amplification (RT-LAMP) to achieve this goal. This novel technique uses a micro-fabricated chip with an array of micro-wells with knife-like sharp well edges to assist in division of a tissue cryosection into small pieces in a process we call "tissue pixelation". The array consists of over five thousand 100um (side length) pyramidal wells with a volume of ~ 175pL each. Following the above tissue transfer step, reagents are loaded onto the chip into the individual wells using a parallel loading process and independent picoliter volume RT-LAMP reactions are performed in each well. Towards this end, we have completed and characterized the chip fabrication, tissue pixelation and picoliter volume reagent loading on chip steps. In addition to this, we have also designed and characterized a novel RT-LAMP reaction for topoisomerase II alpha (TOP2A) mRNA biomarker for LNCaP prostate cancer cells.