Rapid, localized changes in gene expression require mRNA extraction at high temporal and spatial resolution. Current small-scale mRNA extractions depend on the removal of the cells/tissue from an organism or preserved specimens. What these methods have in common is that they are destructive and do not distinguish between genomic DNA and RNA. Therefore, extracted (m)RNA is typically contaminated by extracted cytoplasm, nuclear DNA, or other compounds, and the required purification leads to loss of especially low-abundant mRNA. The need to repeatedly remove mRNA from living material has led to the development of solid phase gene extraction (SPGE). SPGE sampling can be achieved using gene-specific or generic sequences and is not species-specific. Here we demonstrate the versatility and validity of this novel RNA extraction by simultaneously profiling nanos and bicoid mRNA in individual Drosophila eggs. The SPGE technique detects previously described distribution profiles of nanos and bicoid. Its low impact is underscored by the normal development of repeatedly sampled eggs. In our study, quantification of actin mRNA in germinating flax seeds linked gene expression to distinct developmental processes. These data demonstrate the universality of SPGE as a simple generic, analytical, and diagnostic procedure.

Introduction

The need to obtain genetic information from biological specimens has led to the development of sophisticated procedures such as laser capture microdissection (1), cell aspiration (2), and microfluidics (3). The interest in cell-level manipulation of mRNA is heightened by the application of atomic force microscope cantilevers modified for mRNA extraction (4) and mRNA expression (5). Using considerably less technology, solid phase gene extraction (SPGE) is rapid and can be performed in less than 2 min from extraction to reverse transcription. This technique minimizes contamination and does not require additional purification of the sample or removal of cells, tissue, or cytoplasm from the organism. SPGE uses glass needles that are surface-coated with oligo(-dT) or gene-specific oligonucleotide sequences. mRNA is extracted from an individual cell or tissue by direct hybridization to the probe and results in the removal of a representative mRNA sample. The sampled cells remain alive and may be sampled repeatedly at different times or locations. The hybridized mRNA is reverse-transcribed and the amount of generated cDNA is sufficient for multiple real-time PCR analyses using SYBR Green or other reporter systems without pre-amplification.

To corroborate our method, we studied the mRNA extraction from Drosophila eggs and analyzed gene expression in embryonic plant roots. The distribution of nanos and bicoid mRNA in Drosophila eggs has been investigated extensively over the last decades. During oogenesis nanos and bicoid mRNA are derived from maternal nurse cells. Nanos mRNA accumulates in the posterior and bicoid mRNA accumulates in the anterior pole of the egg (6,7). This distribution persists until 2 h after fertilization (8,9). The translation of nanos and bicoid mRNA establishes protein gradients that determine the body axis and the formation of the head and abdomen (6,7).

We sampled individual eggs at four equidistant positions from anterior to posterior and simultaneously determined the absolute copy number of nanos and bicoid mRNA at the time of extraction using a SYBR Green–based (Power SYBR Green Master Mix; Applied Biosystems, Foster City, CA, USA) real-time PCR assay.

To confirm that SPGE is equally applicable to plant systems, we studied the onset of actin gene expression in germinating flax (Linum usitatissimum) seeds. Germination is initiated by imbibition and comprises radicle emergence and subsequent growth of the embryo.

Because necessary mRNAs are present in the seed (10,11), plants do not require transcription for initial germination. However, transcription is essential for their subsequent growth and development (10). Actins are essential proteins and polymerize to filaments (f-actin) that control cellular processes such as vesicle trafficking (12) and cell elongation (13). The expression of actin mRNA isoforms depends on different developmental stages of the embryo during germination (14). We studied the expression of two actin genes (ACT1 and ACT2) in germinating flax roots during imbibition and early germination.

All experiments demonstrate that SPGE is ideally suited to investigate gene expression with high sensitivity. Moreover, SPGE overcomes current limitations of spatial and temporal gene expression profiling.

Materials and Methods

Extraction Probes

Glass needles were pulled on a Sutter P-2000 micropipette puller (Sutter Instruments, Norato, CA, USA) from 1 mm diameter solid borosilicate glass rods (World Precision Instruments, Sarasota, FL, USA) that produced consistent tip diameters of 0.5 to 1 µm as indicated by scanning electron microscopy (SEM). Coating of the needles was based on a modified protocol (15). The glass needles were incubated overnight in 30% ammonium hydroxide, rinsed with distilled water, and incubated for 30 min in a mixture of 1% trimethylmercaptosilane in 95% EtOH. After silanization, the needles were rinsed in 100% EtOH and dried for 2 hat 150°C.

The needles were coated by 30 min incubation in thiol-modified oligo-dT₁₀ nucleotides in 500 mM sodium carbonate buffer (pH 9). The nanos UTR-specific sequence (5′-TCACAGAAACAGACATAAAT-3′; T_m 46.6°C) was amino-linked to 1% aminopropyl-trimethoxysilane-coated needles. All oligonucleotides were purchased from IDT (Coralville, IA, USA). Needles were rinsed in distilled sterile water and stored at room temperature (RT). Storage for three days resulted in no detectable decrease of efficiency.

Needle Characterization

Coating density, hybridization kinetics, and release of mRNA from the needles were tested by incubating the needles in flax poly(A) mRNA that was isolated from 2-day-old flax roots using the Straight A's mRNA Isolation kit (Novagen, Madison, WI, USA) according to the manufacturer's protocol. The mRNA was quantified (1 µl on a Nanodrop ND-1000 spectrophotometer; NanoDrop Technologies, Wilmington, DE, USA), adjusted to 50 µg mRNA × mL⁻¹, and stored at −20°C. For individual experiments this mRNA stock was further diluted as indicated.